Novel method for the production of hybrid maize seeds

ABSTRACT

The invention relates to a method for production and multiplication of maize plants homozygous for a transgene which confers male sterility, useful in the production of hybrid maize seed.

The present invention relates to a method for the production andmultiplication of young maize plants homozygous for a transgeneconferring male sterility, that are useful for the production of hybridmaize seeds.

The production of hybrids via “sexual hybridization” of parents havingparents having different genetic backgrounds is of great importance inmodern agricultural practice. In fact, crossing between plants that areof the same species but not related produces a lineage which manifestscharacteristics, such as yield or disease resistance, greater than thoseof the parents: this is the heterosis or hybrid vigor effect. Thus, theproduction of hybrids is often used to improve both the quality and theyield of crop plants.

In addition, these descendants are genetically uniform, in particular interms of the characteristics of productivity, sensitivity to torrentialrain and to drought, initial vigor and sensitivity to diseases and topests, and are therefore advantageous for farmers.

Without human intervention, the production of hybrids is limited byphenomena of self-fertilization. The production of hybrid seedstherefore makes it necessary to favor cross-fertilization by preventingself-pollination by mechanical, chemical or genetic techniques. Thevarious approaches for preventing self-fertilization by controllingpollination include in particular:

-   -   manual or mechanical “castration” or chemical inactivation of        the male organs of the plant,    -   the use of a modified plant exhibiting recessive nuclear male        sterility,    -   the use of a modified plant exhibiting recessive cytoplasmic        male sterility,    -   the use of a dominant nuclear male sterile plant (artificial        male sterility or AMS).

However, some of these techniques are not completely effective andsometimes even difficult to implement.

-   -   “Castration” is expensive and can cause losses in yield in the        event of error.

The effectiveness of using chemical agents, which is less expensive,depends on the environmental conditions at the time of application ofthe gametocide, which leads the seed producer to take considerable riskseach season.

The use of a recessive nuclear male sterile plant is not easy toimplement, in particular due to the maintaining of the male sterilecharacteristics, which requires reselection of male sterile plants inthe descendents obtained by self-fertilization of a plant heterozygousfor the recessive male sterility gene. To be effective, this systemtherefore involves the use of a marker that is closely linked to themale sterility gene and readily identifiable.

Male sterility can be “acquired”, i.e. it is independent of any geneticmanipulation via the recombinant DNA process. Cytoplasmic male sterilitycan be distinguished from nuclear male sterility.

“Cytoplasmic” male sterility (“CMS”) is related to changes in theorganization and the expression of the mitochondrial genome, nuclearmale sterility results from mutations in the genome of the cell'snucleus. However, CMS is not complete, {fraction (2/1000)} of the plantsremaining fertile. The loss of cytoplasmic genetic diversity whenselectors use the same cytoplasm in their selection program can,moreover, be handicapped.

Male sterility can be “artificial” (“AMS”), i.e. it is induced by theexpression of a gene conferring male sterility (AMS gene) which isinserted either into the mitochondrial genome (cytoplasmic malesterility) or into the nuclear genome (nuclear male sterility).

The AMS system makes it possible to avoid the problems associated withthe other methods. As a result, unlike CMS, the AMS system does notdepend on the existence of a mutant. Maintenance of the sterilecharacteristic of the male line can be obtained using a dominant malesterility gene linked to a marker gene which allows the selection of theartificial male sterile plants.

The problem of maintaining the male sterile characteristic in a plantline has been partly solved in patent application WO 95/34634, whichenvisions crossing between a plant heterozygous for a dominant malesterility gene and a plant which restores fertility, and then selectingthe male sterile seeds. In that application, the use of a marker, linkedto the fertility-restoring gene, which influences the regulation of thegenes encoding the regulatory enzymes for anthocyanin synthesis, makesit possible to differentiate, by means of the color, the male sterileseeds from the male fertile seeds.

However, the separating system described in that patent application doesnot make it possible to reliably separate hybrid seeds on a large scale.

In addition, the general application of this technology is limitedinsofar as the choice of the coloration marker is conditioned by thegenotype of the plant containing the fertility-restoring gene. Inparticular, only plants whose genotype does not condition a visibleproduction of anthocyanin in the seeds can be used.

The present invention therefore proposes a method for the production andmultiplication of young maize plants homozygous for a transgeneconferring artificial nuclear male sterility, allowing hybrid seeds tobe produced, and readily applicable on a large scale. An advantage ofthe method proposed is that its application is not conditioned by thegenotype of the plants used.

This approach is based in particular on combining the fertilitycharacteristic with a “small seed” phenotype. The “small seed” phenotypeis preferentially obtained by the expression of the shrunken 2 andbrittle 2 genes in antisense orientation. The brittle 2 gene encodes oneof the 2 subunits of ADP glucose pyrophosphorylase, an enzyme involvedin starch synthesis. The fragment was obtained by the RT-PCR techniquefrom a maize ear total RNA extract and according to the Genbank data(accession No. S72425). This fragment represents an incomplete cDNA ofthe brittle 2 gene. The shrunken 2 gene encodes the other subunit of ADPglucose pyrophosphorylase. The fragment was obtained by the RT-PCRtechnique from a maize ear total RNA extract and according to theGenbank data (accession No. S72425). This fragment contains the completesequence of the coding region of the shrunken 2 gene.

The inhibition of these two genes for wrinkled mutants of reduced sizeand weight makes it possible to obtain maize seeds of reduced densityand/or size. Such seeds can be readily separated by sieving and/ordensimetric separation, for example using a densimetric table or column,or by flotation, which makes it possible to use a reliable, simpleselection that is automated and therefore applicable on a large scale.

In the context of the present invention, the expression “heterozygousfor the AMS transgene” denotes a young maize plant made male sterile byincorporation into its genome of a single copy of a transgene conferringartificial nuclear male sterility (“AMS”). Unless otherwise indicated,this young plant does not contain the fertility-restoring gene linked toa “small seed” phenotype marker.

In the context of the present invention, the expression “homozygous forthe AMS transgene” denotes a young maize plant made male sterile byincorporation into its genome two copies, located at the same place oneach of the sister chromosomes, of a transgene conferring artificalnuclear male sterility (AMS). Unless otherwise indicated, this youngplant does not contain the fertility-restoring gene linked to a “smallseed” phenotype marker.

The expression “fertility-restoring young maize plant comprising in itsgenome a fertility-restoring gene linked to a “small seed” phenotypemarker” is intended to mean a young maize plant heterozygous orhomozygous for said fertility-restoring gene linked to a “small seed”phenotype marker. Preferably, said young maize plant is heterozygous.Unless otherwise indicated, this young plant does not contain atransgene conferring artificial nuclear male sterility (AMS).

The expression “young maize plant comprising in its genome an AMStransgene” is intended to mean a young maize plant heterozygous orhomozygous for said AMS transgene.

The expression “young maize plant having a wild-type genotype” isintended to mean a young maize plant which contains in its genomeneither a transgene conferring artificial nuclear male sterility (AMS)nor a fertility-restoring gene linked to a “small seed” phenotypemarker. Preferably, said young maize plant having a wild-type genotypebelongs to an elite line.

The expression “small seed” phenotype is intended to mean seeds forwhich the density and/or the size and/or the mass is (are) smaller thanthat of a seed of normal size, preferably from 40 to 50% smaller. In thecontext of the present invention, these seeds may be referred to as“deficient seeds” or “wrinkled seeds”.

The expression “small seed phenotype marker” or “gene which confers asmall seed phenotype” or “gene encoding a small seed phenotype” isintended to mean any gene, in the sense orientation or in the antisenseorientation, which, when it is expressed in the plant, confers a “smallseed” phenotype. Among these markers mention may in particular be madeof the shrunken 2, brittle 2 and shrunken 1 genes and the miniaturelocus 1 which encodes an invertase; and, more generally, any gene whichmakes it possible to decrease the starch content and does not impair theseed viability.

The expression “seed of normal size” or “normal seed” or “seed of normalphenotype” is intended in particular to mean a seed whose size and/ordensity and/or mass has or have not been modified, in particular byintegration into the genome of the plant of a “small seed” phenotypemarker.

The term “elite line” is intended to mean a line having a substantialagronomic and commercial potential, at a given period.

The term “linkage” or “genetic linkage” is intended to mean a geneticdistance sufficiently small for the frequencies of recombination duringmeiosis to be negligible.

The term “protein of interest” is intended to mean a protein of human oranimal origin which may be of therapeutic and/or prophylactic interest,such as collagen, gastric lipase, antibodies, etc.

One of the aims of the invention is therefore to propose a method forthe production of hybrid maize seeds by crossing a young maize plantheterozygous for a transgene conferring artificial nuclear malesterility (“AMS”) with a young maize plant having a wild-type genotype.The system according to the present invention, which advantageouslymakes it possible to maintain the male sterile characteristic of youngmaize plants, is applicable independently of the genotype of the youngmaize plants used.

The solution provided by the invention consists in initially producingmaize seeds homozygous for a transgene conferring artificial nuclearmale sterility (“AMS”) and heterozygous for a fertility-restoring genelinked to a “small seed” phenotype marker. Using these seeds, selectorscan easily introgress the genotype homozygous for the AMS transgene andheterozygous for a fertility-restoring gene linked to a “small seed”phenotype marker into a maize line having a wild-type genotype, and inparticular into an elite line of interest, by successive backcrosses. Aconverted elite line homozygous for a transgene conferring artificialnuclear male sterility (“AMS”) and heterozygous for afertility-restoring gene linked to a “small seed” phenotype marker canthus be obtained. Self-fertilization of young maize plants derived fromthese seeds then makes it possible to produce:

-   -   seeds homozygous for the AMS transgene generating male sterile        young maize plants which, when crossed with young maize plants        having a wild-type genotype, and in particular young plants        belonging to an elite line used for conversion, where        appropriate, will produce seeds heterozygous for the AMS        transgene. Fertilization of male sterile young maize plants        derived from these seeds, by an unrelated young maize plant        having a wild-type genotype then produces hybrid seeds which        benefit from the vigor effect (heterosis),    -   seeds homozygous for the AMS transgene and heterozygous or        homozygous for the fertility-restoring gene linked to a “small        seed” phenotype marker, generating non-male sterile young maize        plants, the self-fertilization of which produces in particular        seeds homozygous for the AMS transgene and heterozygous for the        fertility-restoring gene linked to a “small seed” phenotype        marker, thus ensuring renewal of the male sterile young maize        plants of interest.

Advantageously, when the AMS transgene is genetically linked to a geneencoding a protein of interest, the hybrid seeds homozygous for the AMStransgene are found to be particularly useful for generating young maizeplants producing said protein of interest.

In the context of the present invention, selection of the maize seeds bymeans of the “small seed” phenotype can in particular be carried out bya method of size separation or densimetric separation.

A size separation can be carried out so as to separate the seeds as afunction of their length, using for example an indent separator, oftheir width, with for example a disk separator, or of their thickness,using for example a calibrator.

The principle of densimetric separation is based on separation of theseeds according to their density and uses in particular a densimetriccolumn or a densimetric table, or a flotation system.

More particularly,

-   -   the indent separator is made up of a rotating horizontal        cylinder. The separation is carried out by centrifugal force.        The smallest seeds enter into the indents (which cover the        inside of the cylinder) and are kept there by the centrifugal        force;    -   the disk separator is made up of thick disks arranged vertically        with indents of appropriate size hollowed out in their        thickness;    -   the most conventional calibrators are thickness-calibrators made        up of flat or cylindrical sieves with elongated perforations        which force the seed to present itself according to its smallest        thickness in order to pass through. The round-perforation        calibrator works on the same principle, but the round orifices        with small “notches” inside the cylinder forcing the seed to        present itself vertically in order to pass through the sieve;    -   the densimetric column comprises a vibrating distributor which        introduces the mixture of seeds to be separated halfway up a        hollow column in which there is an ascending homogeneous stream        of air. The heavy particles fall whereas the lighter particles        rise. The separation is thus effected;    -   a densimetric table makes it possible to separate bodies of the        same size but with a different specific weight. The specific        weight corresponds to a measurement of the mass of a quantity of        seeds relative to its volume. The principle of the device is a        workstation (often consisting of a lattice made of metal wire or        textile) through which a uniform air stream passes, which        fluidifies the mixture of seeds and causes the stratification        thereof schematically into two layers. The heavy products remain        close to the table and the light products above. Separation of        the two layers is obtained by adjusting the incline of the        workstation (in two directions) and by a transverse agitating        movement backward and forward. A densimetric table such as that        sold by the company Cimbria Heid GmbH (Stockerau, Austria) may,        for example, be used;    -   the flotation system is a system which makes it possible to        separate the seeds as a function of their density and/or mass,        based on their ability to float on a liquid, in particular        water. The heaviest seeds (having the least ability to float)        are found at the bottom and the lightest seeds (having the        greatest ability to float) are found at the surface of the        liquid, which enables the separation. When this system is used,        the seeds should remain in contact with the water for as little        time as possible, so as not to impair their germination        capacity. A drying step may sometimes be necessary.

The present invention therefore proposes a method for the production ofmaize seeds homozygous for a transgene conferring artificial nuclearmale sterility (“AMS”) and heterozygous for a fertility-restoring genelinked to a “small seed” phenotype marker, comprising the stepsconsisting in:

-   -   a) crossing a male sterile young maize plant heterozygous for        the AMS transgene with a fertility-restoring young maize plant        comprising in its genome a fertility-restoring gene linked to a        “small seed” phenotype marker,    -   b) selecting, by means of the “small seed” phenotype, the maize        seeds comprising in their genome a fertility-restoring gene        linked to a “small seed” phenotype marker,    -   c) self-fertilizing the young maize plants derived from seeds        selected according to step b),    -   d) selecting the seeds homozygous for the AMS transgene and        heterozygous for the fertility-restoring gene linked to a “small        seed” phenotype marker.

Preferably, at least one selection step in the method comprisesdensimetric separation, in particular using a densimetric table orcolumn, or a flotation system.

The selection step thus carried out has the advantage of allowing aseparation of the seeds, as a function of their “small seed” or “normal”phenotype, which can be readily implemented and automated.

According to a particular embodiment, step b) of the method describedabove may advantageously be replaced with a genotyping step. The presentinvention therefore also proposes a method for the production of maizeseeds homozygous for a transgene conferring artificial nuclear malesterility (“AMS”) and heterozygous for a fertility-restoring gene linkedto a “small seed” phenotype marker, comprising the steps consisting in:

-   -   a) crossing a male sterile young maize plant heterozygous for        the AMS transgene with a fertility-restoring young maize plant        comprising in its genome a fertility-restoring gene linked to a        “small seed” phenotype marker,    -   b) genotyping the seeds obtained by means of the cross according        to step a), and selecting the maize seeds comprising in their        genome a fertility-restoring gene linked to a “small seed”        phenotype marker,    -   c) self-fertilizing the young maize plants derived from the        seeds genotyped according to step b),    -   d) selecting the seeds homozygous for the AMS transgene and        heterozygous for the fertility-restoring gene linked to a “small        seed” phenotype marker.

According to a particular embodiment, said young maize plantheterozygous for a transgene conferring artificial nuclear malesterility also contains a gene encoding a protein of interest,preferably genetically linked to the AMS transgene.

The implementation of the methods described above makes it possible toproduce a maize seed homozygous for an AMS transgene and heterozygousfor a fertility-restoring gene linked to a “small seed” phenotypemarker. The present invention is therefore directed toward a maize seedhomozygous for an AMS transgene and heterozygous for afertility-restoring gene linked to a “small seed” phenotype marker,which can be obtained by one of the above methods. Preferably, thegenotype that is AMS-homozygous and heterozygous for afertility-restoring gene linked to a “small seed” phenotype marker canbe introgressed into a maize line having a wild-type genotype, and inparticular into an elite line of interest, by successive backcrosses.

The cultivating, and then the self-fertilizing of the young maize plantsobtained from seeds above (homozygous for an AMS transgene andheterozygous for a fertility-restoring gene linked to a “small seed”phenotype marker) produces maize seeds homozygous for the AMS transgeneand also maize genes homozygous for the AMS transgene and heterozygousor homozygous for the fertility-restoring gene linked to a “small seed”phenotype marker. The maize seeds homozygous for the AMS transgene canbe readily differentiated from the other seeds thus produced since onlythey have seeds of normal phenotype.

The present invention therefore proposes a method for the production ofmaize seeds homozygous for a transgene conferring artificial nuclearmale sterility (“AMS”), comprising the steps consisting in:

-   -   a) self-fertilizing the young maize plants derived from seeds        homozygous for the AMS transgene and heterozygous for a        fertility-restoring gene linked to a “small seed” phenotype        marker, which can be obtained by one of the methods described        above,    -   b) selecting seeds homozygous for the AMS transgene.

Preferably, the selection step comprises densimetric separation, inparticular using a densimetric table or column, or a flotation system.This step advantageously makes it possible to separate the seedshomozygous for the AMS transgene by means of their normal seedphenotype.

According to another embodiment, the method for the production of maizeseeds homozygous for a transgene conferring artificial nuclear malesterility (“AMS”), comprises the steps consisting in:

-   -   a) crossing a male sterile young maize plant heterozygous for        the AMS transgene with a fertility-restoring young maize plant        comprising in its genome a fertility-restoring gene linked to a        “small seed” phenotype marker,    -   b) selecting, by means of the “small seed” phenotype, the maize        seeds comprising in their genome a fertility-restoring gene        linked to a “small seed” phenotype marker,    -   c) self-fertilizing the young maize plants derived from the        seeds selected according to step b),    -   d) selecting seeds homozygous for the AMS transgene and        heterozygous for the fertility-restoring gene linked to a “small        seed” phenotype marker,    -   e) self-fertilizing young maize plants derived from seeds        according to step d),    -   f) selecting seeds homozygous for the AMS transgene.

According to a particular embodiment, said young maize plantheterozygous for a transgene conferring artificial nuclear malesterility also contains a gene encoding a protein of interest,preferably genetically linked to the AMS transgene.

Step e) of this method may optionally be preceded by a step ofsuccessive backcrosses with a young maize plant having a wild-typegenotype, and in particular a young maize plant belonging to an eliteline, so as to convert this young maize having a wild-type genotype withthe genotype homozygous for the AMS transgene and heterozygous for thefertility-restoring gene linked to a “small seed” phenotype marker. Theseeds derived from this backcrossing step are then used for thecontinuation of the method.

Preferably, at least one selection step, and in particular step f),comprises densimetric separation, in particular using a densimetrictable or column, or a flotation system. This step advantageously makesit possible to separate the seeds homozygous for the AMS transgene bymeans of their normal seed phenotype.

According to another variant, step b) of the method above is replacedwith a genotyping step. The present invention therefore also relates toa method for the production of maize seeds homozygous for a transgeneconferring artificial nuclear male sterility (“AMS”), comprising thesteps consisting in:

-   -   a) crossing a male sterile young maize plant heterozygous for        the AMS transgene with a fertility-restoring young maize plant        comprising in its genome a fertility-restoring gene linked to a        “small seed” phenotype marker,    -   b) genotyping the seeds obtained by means of the cross according        to step a), and selecting the maize seeds comprising in their        genome a fertility-restoring gene linked to a “small seed”        marker,    -   c) self-fertilizing the young maize plants derived from the        seeds genotyped according to step b),    -   d) selecting the seeds homozygous for the AMS transgene and        heterozygous for the fertility-restoring gene linked to a “small        seed” phenotype marker,    -   e) self-fertilizing young maize plants derived from seeds        according to step d),    -   f) selecting seeds homozygous for the AMS transgene.

According to a particular embodiment, said young maize plantheterozygous for a transgene conferring artificial nuclear malesterility also contains a gene encoding a protein of interest,preferably genetically linked to the AMS transgene.

Step e) of this method may optionally be preceded by a step ofsuccessive backcrosses with a young maize plant having a wild-typegenotype, and in particular a young maize plant belonging to an eliteline, so as to convert this young maize plant having a wild-typegenotype with the genotype homozygous for the AMS transgene andheterozygous for the fertility-restoring gene linked to a “small seed”phenotype marker. The seeds derived from this backcrossing step are thenused for the continuation of the method.

Preferably, at least one selection step and in particular step f),comprises densimetric separation, in particular using a densimetic tableor column, or a flotation system. This step advantageously makes itpossible to separate the seeds homozygous for the AMS transgene by meansof their normal seed phenotype.

The cultivating of seeds homozygous for the AMS transgene then makes itpossible to generate male sterile young maize plants which, when crossedwith young plants having a wild-type genotype, in particular with youngplants belonging to an elite line, produce seeds heterozygous for theAMS transgene. The elite line used for this cross may in particular bethat used for the backcrossing step optionally carried out in themethods above. In this case, the seeds produced can be used to producecommercial seeds derived from a maize hybrid. According to anotherembodiment, the wild-type elite line can be replaced with a maize hybridso as to obtain seeds which will serve to produce a three-way hybrid.

According to another aspect of the invention, the method for theproduction of maize seeds homozygous for the AMS transgene can be usedfor the production of proteins of interest, in particular therapeuticand/or prophylactic interest. The protein of interest may be producedthroughout the plant or else preferentially concentrated in specificorgans, such as the seeds.

The method according to the invention has the advantage of making itpossible to produce such proteins under controlled and large-scaleconditions. It is therefore of great value to the pharmaceutical field.

To do this, a vector containing the barnase gene, conferring malesterility, under the control of the A9 promoter, and a gene oftherapeutic and/or prophylactic interest can be constructed. This vectorcan be used to obtain plants containing the gene conferring malesterility and the gene of therapeutic and/or prophylactic interest,which may then be used according to the system of production asdescribed in order to produce seeds expressing the protein oftherapeutic and/or prophylactic interest.

In particular, such a vector can be used to obtain a young maize plantheterozygous for an AMS transgene (and therefore for the gene encodingthe protein of interest), which young maize plant can then be crossedwith a fertility-restoring young maize plant of genotype (+/+; SSB/+).

Advantageously, the gene of therapeutic and/or prophylactic interest isgenetically linked to the AMS transgene.

Such a system makes it possible to produce only the protein oftherapeutic and/or prophylactic interest in the seed, for example, or inan organ of the plant, whatever the developmental stage, depending onthe regulatory sequence used in the gene encoding said protein ofinterest, since the protein conferring male sterility is not produced inthe organs of the plant. This system thus makes it possible to avoiddissemination of the transgene of therapeutic and/or prophylacticinterest via the pollen, since the plants are completely male sterile.

The present invention therefore also relates to a method for theproduction of a seed heterozygous for an AMS transgene, comprising thecrossing of a young maize plant derived from a seed homozygous for anAMS transgene, which can be obtained by one of the methods for theproduction of a seed homozygous for the AMS transgene described above,with a young maize plant having a wild-type genotype.

According to another embodiment, the method for the production of a seedheterozygous for an AMS transgene is characterized in that one of themethods for the production of a seed homozygous for the AMS transgenedescribed above also comprises the crossing of a young maize plantderived from said seed homozygous for an AMS transgene with a youngmaize plant having a wild-type genotype.

Fertilization of male sterile young maize plants derived from theseseeds, by an unrelated young maize plant having a wild-type genotype,advantageously produces hybrid seeds which benefit from the vigor effect(heterosis).

According to another aspect, the system proposed by the presentinvention advantageously makes it possible to maintain young maizeplants homozygous for an AMS transgene and heterozygous for afertility-restoring gene linked to a “small seed” phenotype marker. Themethods for the production of maize seeds homozygous for the AMStransgene described above in fact comprise a final selection of seeds,in particular on the basis of their normal seed phenotype. The seeds of“small seed” phenotype not selected by these methods can be sown, andthe young plants generated can then be self-fertilized, thus producingin particular seeds homozygous for the AMS transgene and heterozygousfor the fertility-restoring gene linked to a “small seed” phenotypemarker.

The invention therefore relates to a method for the multiplication of ayoung maize plant homozygous for an AMS transgene and heterozygous for afertility-restoring gene linked to a “small seed” phenotype marker,comprising the steps consisting in:

-   -   a) self-fertilizing young maize plants homozygous for an AMS        transgene and heterozygous for a fertility-restoring gene linked        to a “small seed” phenotype marker, which can be obtained from a        seed from one of the methods for the production of seeds        homozygous for an AMS transgene and heterozygous for a        fertility-restoring gene linked to a “small seed” phenotype        marker described above,    -   b) selecting seeds homozygous for the AMS transgene and having a        “small seed” phenotype,    -   c) selecting the seeds homozygous for the AMS transgene and        heterozygous for a fertility-restoring gene linked to a “small        seed” phenotype marker, obtained by self-fertilization of the        young maize plants obtained from the seeds obtained according to        step b).

Preferably, step b) of the method above comprises densimetricseparation, in particular using a densimetric table or column, or aflotation system. A subject of the present invention is also nucleotideconstructs, referred to as expression cassettes, comprising a promoternucleotide sequence functionally linked to at least one gene ofinterest.

Said gene of interest may also be combined with other regulatoryelements such as activators and transcription termination sequences(terminators). Other elements such as introns, enhancers,polyadenylation sequences and derivatives can also be present in thenucleic acid sequence of interest, in order to improve the expression orthe functioning of the transforming gene. The expression cassette mayalso contain 5′ untranslated sequences referred as “leader” sequences.Such sequences can improve translation.

Preferably, in the methods for the production of seeds described above,the young maize plant comprising an AMS transgene conferring artificialnuclear male sterility is characterized in that the AMS transgene,preferably the barnase gene (Hartley, 1988; Gene Bank No. X 12871), isincluded in an expression cassette, under the control of a promoterspecific for pollen generation and of a terminator, and geneticallylinked to a gene encoding a selection agent under the control of apromoter and of a terminator.

Advantageously, said expression cassette may also comprise a geneencoding a protein of interest.

Preferably, said gene encoding a protein of interest is geneticallylinked to the AMS gene, in particular the barnase gene. More preferably,the gene encoding a protein of interest is not under the control of saidpromoter specific for pollen generation.

The promoter specific for pollen generation is in particular a promoterwhich allows specific expression in the anther, chosen from the groupconsisting of the A3 promoter (WO 92/11379), the A6 promoter, the A9promoter (WO 92/11379), corresponding to the 5′ noncoding region of theArabidopsis thaliana A9 gene, and the anther tapetum-specific promoterssuch as TA29, TA26, TA13 (WO 89/10396) or Mac2 (WO 00/68403). Among thegenes encoding a selection agent (also referred to as selection markergenes), use may in particular be made of genes which confer resistanceto an antibiotic (Herrera-Estrella et al., 1983) such as hygromycin,kanamycin, bleomycin or streptomycin, or to herbicides (EP 242 246),such as glufosinate, glyphosate or bromoxynil.

Preferably, said gene encoding a selection agent is chosen from the bargene (White et al., 1990; Gene Bank No. X 17220) which confersresistance to the herbicide Basta® (glufosinate) and the NptII genewhich confers resistance to kanamycin (Bevan et al., 1983).

The excision system for removing the gene encoding a selection agent maybe a transposition system, such as in particular the maize Ac/Ds system(WO 02/101061), or a recombination system, such as in particular the P1bacteriophage Cre/lox system, the yeast FLP/FRT system (Lyzrik et al.,1997), the Mu phage Gin recombinase, the E. coli Pin recombinase or thepSR1 plasmid R/RS system. A cotransformation system (Komari et al.,1996) can also be used. Preferably, the system used will be the maizeAc/Ds system.

According to a preferred embodiment, said gene is included within the Dstransposable element (also called dissociating element or mobilizablesequence of a transposon).

The Ds transposon used is described in the publication by Yoder et al.(1993). The Ds element is an Ac element which has undergone importantmutations or deletions in the sequence encoding the transposase. It isable to excise itself from its insertion site only in the presence of anactive transposase source Ac. It is therefore Ac-dependent. A preferredsystem for removing a gene encoding a selection agent can comprise twocomponents:

-   -   a first plant having no active transposase, into which a        construct comprising the cassette for expression of the gene of        interest and that of the gene encoding a selection agent,        bordered by the mobilizable sequences of a transposon, can be        integrated,    -   a second plant containing in its genome a gene encoding an        endogenous active transposase.

The crossing of these two plants results in the obtaining ofregenerants, obtained from F1 plants or from F2 plants selected for thepresence of the gene of interest but no gene encoding a selection agent.

Preferably according to the invention, the promoter combined with thegene encoding a selection agent is a constitutive promoter, such as theactin promoter-actinintron, corresponding to the 5′ noncoding region ofthe rice actin 1 gene and its first intron (McElroy et al., 1991; GeneBank No. S 44221). The presence of the first actin intron makes itpossible to increase the level of expression of a gene when it is fused3′ of a promoter. This promoter sequence allows, for example,constitutive expression of the bar gene.

Among the terminators which may be used with the AMS transgene or thegene encoding a selection agent, mention may in particular be made of:

-   -   the Nos 3′ terminator, nopaline synthase terminator which        corresponds to the 3′ noncoding region of the nopaline synthase        gene originating from the Ti plasmid of Agrobacterium        tumefaciens nopaline strain (Depicker et al., 1982), and    -   the CaMV 3′ terminator, corresponding to the 3′ noncoding region        of the sequence of the circular double-stranded DNA cauliflower        mosaic virus producing the 35S transcript (Franck et al. 1980;        Gene Bank No. V 00141).

According to another aspect, the present invention relates to anexpression cassette comprising a fertility-restoring gene geneticallylinked to at least one gene encoding a “small seed” phenotype, combinedwith elements which allow their expression in plant cells, in particulara transcription promoter and terminator.

Among the transcription promoters which can be used in combination withthe gene encoding a “small seed” phenotype, mention may be made inparticular of:

-   -   the HMWG (high molecular weight glutenin) promoter corresponding        to the 5′ noncoding region of the wheat (Triticum aestivum)        glutenin gene, an albumen storage protein. This seed-specific        promoter is described in the publication by Robert et al.        (1989),    -   the B32 promoter, described in the publication by N. Di Fonzo et        al. (1988).

Preferably, said expression cassette comprising a fertility-restoringgene genetically linked to at least one gene encoding a “small seed”phenotype is characterized in that said fertility-restoring gene is thebarstar gene (Hartley, 1998) placed under the control of a promoterspecific for pollen generation, in particular an anther-specificpromoter such as pA3, pA6, pA9 or pTA29, or of the Mac2 promoter, and ofthe CaMV 3′ terminator or Nos 3′ terminator, genetically linked to agene encoding a selection agent under the control of the actinpromoter-actin intron and of the CaMV 3′ terminator or Nos 3′terminator.

Among the genes encoding a selection agent, use may in particular bemade of genes which confer resistance to an antibiotic such ashygromycin, kanamycin, bleomycin or streptomycin, or to herbicides suchas glufosinate, glyphosate or bromoxynil. Preferably, said gene encodinga selection agent is chosen from the bar gene which confers resistanceto the herbicide Basta® and the NptII gene which confers resistance tokanamycin.

Preferably, the gene encoding a “small seed” phenotype is chosen fromthe shrunken 2 and brittle 2 genes in antisense orientation.

According to another aspect, the invention relates to a vector, inparticular a plasmid, characterized in that it contains at least oneexpression cassette as described above.

The invention also relates to a cellular host, in particular a bacteriumsuch as Agrobacterium tumefaciens, transformed with said vector. Such acellular host is used for transfecting maize cells with a vectoraccording to the invention.

The invention therefore also relates to a maize cell transformed with atleast one vector as described above. The transformation of plant cellscan be carried out by transfer of the abovementioned vectors into theprotoplasts, in particular after incubation of the latter in a solutionof polyethylene glycol (PG) in the presence of divalent cations (Ca²⁺)according to the method described in the article by Krens et al. (1982).

The transformation of the plant cells can also be carried out byelectroporation, in particular according to the method described in thearticle by Fromm et al. (1986).

The transformation of the plant cells can also be carried out using agene gun for projecting, at very high speed, metal particles coveredwith DNA sequences of interest, thus delivering genes into the cellnucleus, in particular according to the technique described in thearticle by Finer et al. (1992).

Another method for transforming the plant cells is that of cytoplasmicor nuclear microinjection.

According to a particularly preferred embodiment of the method of theinvention, the plant cells are transformed with a vector according tothe invention, said cellular host being capable of infecting said plantcells, allowing the integration into the genome of the latter of the DNAsequences of interest initially contained in the genome of theabovementioned vector.

Advantageously, the abovementioned cellular host used is “Agrobacteriumtumefaciens”, in particular according to the methods described in thearticles by Bevan (1984) and by An et al. (1986), or else Agrobacteriumrhizogenes, in particular according to the method described in thearticle by Jouanin et al. (1987).

Preferably, the transformation of the plant cells is carried out bytransfer of the T region of the tumor-inducing extrachromosomal circularplasmid Ti of Agrobacterium tumefaciens, using a binary system (Watsonet al., 1994).

To do this, two vectors are constructed. In one of these vectors, theT-DNA region has been removed by deletion, with the exception of theright and left edges, a marker gene being inserted between them so as toallow selection in the plant cells. The other partner of the binarysystem is an auxiliary Ti plasmid, which is a modified plasmid that nolonger has any T-DNA but still contains the vir virulence genes requiredto transform the plant cell. This plasmid is maintained inAgrobacterium.

An object of the present invention is also to produce transgenic youngmaize plants, parts of a plant or plant extracts, characterized in thatthey are regenerated from the transformed plant cell.

The invention relates in particular to a fertility-restoring young maizeplant, characterized in that it comprises in its genome afertility-restoring gene linked to a “small seed” phenotype marker, or ayoung maize plant homozygous for an AMS transgene and heterozygous for afertility-restoring gene linked to a “small seed” phenotype marker,obtained from a maize seed homozygous for an AMS transgene andheterozygous for a fertility-restoring gene linked to a “small seed”phenotype marker.

The invention is also directed toward a kit for implementing a methodfor the multiplication of a young maize plant homozygous for an AMStransgene and heterozygous for a fertility-restoring gene linked to a“small seed” phenotype marker described above, characterized in that itcomprises maize seeds homozygous for an AMS transgene and heterozygousfor a fertility-restoring gene linked to a “small seed” phenotypemarker, and oligonucleotides specific for the AMS transgene that areuseful as primers for detecting, by PCR, the seeds homozygous for an AMStransgene and heterozygous for a fertility-restoring gene linked to a“small seed” phenotype marker.

The examples and figures below illustrate the invention without howeverlimiting the scope thereof:

FIGURE LEGENDS

FIG. 1 represents the diagram of the principle of a succession of stepsresulting in the production of a hybrid seed according to the invention.

FIG. 2 represents the plasmid pRec 274 comprising the barnase gene,conferring male sterility, and the bar gene, conferring resistance tothe herbicide Basta®.

FIG. 3 represents the donor plasmid pBIOS 274 comprising thespectinomycin-resistance gene and also the T-DNA carrying the barnasegene, under the control of the A9 promoter, and the bar gene, under thecontrol of the rice actin promoter.

FIG. 4 represents the donor plasmid pBIOS 424 comprising the A9promoter-barnase-CaMV 3′ terminator and the kanamycin-resistance geneNptII in a Ds dissociating element.

FIG. 5 represents the plasmid p3222 comprising the antisense sequence ofthe brittle 2 gene and also the fertility-restoring barstar gene.

FIG. 6 represents the plasmid p3223 comprising the antisense sequence ofthe shrunken 2 gene and also the fertility-restoring barstar gene.

FIG. 7 represents the plasmid p4962 comprising the antisense sequencesof the shrunken 2 and brittle 2 genes and also the fertility-restoringbarstar gene.

FIG. 8 represents the plasmid pDM 302 comprising the expression cassettefor the bar gene.

FIG. 9 represents the donor plasmid pBIOS 273 which contains anexpression cassette comprising the rice actin promoter, the bar gene andthe Nos 3′ terminator.

EXAMPLES

A nonlimiting illustration of one of the methods according to thepresent invention is described in FIG. 1.

The construction of the various plasmids and the ligation thereof, andthe transformation of Escherichia coli XLI blue bacteria made competentbeforehand, are carried out by means of the usual recombinant DNAtechniques (Sambrook et al., 1989).

The construction of the expression vectors and the transformationtechniques used are within the scope of those skilled in the artfollowing standard techniques.

Example 1 AMS Construct

1.a) Construction of a Plasmid (pRec 274) Comprising the Barnase Gene,Conferring Male Sterility, Linked to the Bar Gene, Conferring Resistanceto the Herbicide Basta®

The vector used for transforming the maize with Agrobacteriumtumefaciens is in the form of a superbinary plasmid of approximately 50kb (pRec 274).

The superbinary vector used for the transformation contains:

-   -   an ori region: Col E1 plasmid origin of replication, necessary        for maintenance and multiplication of the plasmid Escherichia        coli. This origin of replication is not functional in        Agrobacterium tumefaciens,    -   an origin of replication that is functional in Agrobacterium        tumefaciens and Escherichia coli,    -   the cos region of the lambda bacteriophage, which may be of use        for manipulating the vector in vi tro,    -   the additional virB, virC and virG regions of Agrobacterium        tumefaciens which increase the transformation efficiency,    -   genes for resistance to tetracycline (Tetra) and to        spectinomycin (Spect), which are expressed only in the bacteria,    -   a T-DNA carrying the barnase gene conferring male sterility and        the bar gene conferring resistance to the herbicide Basta®,        these two genes being functionally linked to elements which        allow their transcription. In the present example, the barnase        gene is under the control of the A9 promoter and of the CaMV 3′        terminator, the bar gene being under the control of the rice        actin promoter-actin intron and of the Nos 3′ terminator.

The Streptomyces hygroscopicus bar gene encodes a phosphinothricin acyltransferase (PAT) which detoxifies phosphinothricin (Basta® herbicideselection agent) by acetylation (White et al., 1990). It is generallyused to select transformed plants which contain both the gene ofinterest and this gene encoding a selection agent, and which aretherefore resistant to the herbicide.

The barnase gene, which confers male sterility, encodes a ribonuclease(RNase). This gene was isolated from Bacillus amyloliquefasciens and isdescribed in the publication by Hartley (1988).

The superbinary plasmid pRec 274 is represented in FIG. 2.

This superbinary vector is attained by homologous recombination of anacceptor plasmid pSB1 (EP 672 752), derived from an Agrobacteriumtumefaciens Ti plasmid, with a donor plasmid pBIOS 274 (FIG. 3), derivedfrom pUC (Messing, 1983).

The donor plasmid possesses the spectinomycin-resistance gene and alsothe T-DNA carrying the barnase gene, under the control of the A9promoter, and the bar gene, which is also a selection gene, under thecontrol of the rice actin promoter.

The donor and acceptor plasmids possess a region of homology that issufficient to allow homologous recombination and to obtain the“superbinary” vector.

The technique of transformation with Agrobacterium tumefaciens allowsintegration of only the T-DNA consisting of the right border (RB) andleft border (LB) framing the gene of interest (barnase) and the geneencoding a selection agent.

1.b) Construction of a Plasmid (pRec 424) Comprising the Barnase GeneConferring Male Sterility Linked to the NPT-II Gene Conferring KanamycinResistance

The vector used for transforming the maize with Agrobacteriumtumefaciens is in the form of a superbinary plasmid of approximately 50kb (pRec 424).

The superbinary vector used for the transformation contains:

-   -   an ori region: Col E1 plasmid origin of replication, necessary        for maintenance and multiplication of the plasmid Escherichia        coli. This origin of replication is not functional in        Agrobacterium tumefaciens,    -   an origin of replication that is functional in Agrobacterium        tumefaciens and Escherichia coli,    -   the cos region of the lambda bacteriophage, which may be of use        for manipulating the vector in vitro,    -   the additional virB, virC and virG regions of Agrobacterium        tumefaciens which increase the transformation efficiency,    -   genes for resistance to tetracycline (Tetra) and to        spectinomycin (Spect), which are expressed only in the bacteria,    -   a T-DNA carrying the barnase gene, conferring male sterility,        and NptII gene inserted into a Ds element, conferring kanamycin        resistance, these two genes being functionally linked to        elements which allow their transcription. In the present        example, the barnase gene is under the control of the A9        promoter and of the CaMV 3′ promoter, the NptII gene inserted        into a Ds element being under the control of the actin promoter        and of the Nos 3′ terminator.

The NptII gene was isolated from the Escherichia coli Tn5 transposon(Berg et al., 1983). This gene encodes the neomycin phosphotransferasetype II enzyme which catalyzes the O-phosphorylation of aminoglycosideantibiotics such as neomycin, kanamycin, gentamycin and G418 (Davies andSmith, 1978). This gene confers kanamycin resistance, which is used as aselection agent in plant genetic transformation. It is described byBevan et al. (Genbank No. U00004).

The barnase gene, which confers male sterility, encodes a ribonucleaseas mentioned in Example 1.a above.

This superbinary vector is obtained by homologous recombination of anacceptor plasmid pSB1 (EP 672 752), derived from an Agrobacteriumtumefaciens Ti plasmid, with a donor plasmid pBIOS 424 (FIG. 4), derivedfrom pUC (Messing, 1983).

The donor plasmid pBIOS 424 possesses the spectinomycin-resistance geneand also the T-DNA carrying the barnase gene under the control of the A9promoter and the NptII gene placed under the control of the actinpromoter, inserted into a Ds element.

The donor plasmid pBIOS 424 (A9 promoter-barnase-CaMV 3′ terminator andthe kanamycin-resistance gene NptII in a Ds dissociating element) wasgenerated in the following way:

The fragment containing the A9 promoter-barnase gene-CaMV 3′ terminatorcassette was isolated by:

-   -   a) restriction with XhoI,    -   b) treatment with T4 DNA polymerase so as to generate blunt        ends, and    -   c) restriction with XbaI using the plasmid pBIOS 274 (FIG. 3).

This fragment (XhoI/blunt-XbaI) was then introduced into the vectorpBIOS 415 (described below) opened by means of the EcoRV and XbaIrestriction enzymes.

The EcoRV-XbaI fragment thus generated contains the plasmid sequences ofthe vector pSB12 (Japan Tobacco, EP 672 752) and the cassette Dselement-actin promoter-actin intron-NPTII gene-Nos terminator-Dselement.

The plasmid pBIOS 415 contains the GFP gene under the control of theCsVMV promoter (WO 97/48819) and of the Nos terminator (XbaI-XhoIfragment) in the acceptor vector pBIOS 340. The vector pBIOS 340 is avector containing the plasmid sequences of the vector pSB12 (JapanTobacco, EP 672 752) and the cassette Ds element-actin promoter-actinintron-NPTII gene-Nos terminator-Ds element.

The donor and acceptor plasmids possess a region of homology that issufficient to make it possible to obtain the “superbinary” vector byhomologous recombination.

The technique of transformation with Agrobacterium tumefaciens allowsintegration of only the T-DNA consisting of the right border (RB) andleft border (LB) framing the gene of interest (barnase gene) and thegene encoding a selection agent.

Example 2 Construct Restorer-Phenotypic Marker Linked to Seed Size

2.a) Construction of a Plasmid (p3222, FIG. 5) Comprising the AntisenseSequence of the Brittle 2 Gene and also the Fertility-Restoring BarstarGene

The brittle 2 gene encodes a subunit of ADP glucose pyrophosphorylase,an enzyme involved in starch synthesis. In the antisense orientation, itmakes it possible to inhibit the synthesis of this subunit, whichproduces a mutant phenotype in which the seed size is 50% less than thenormal size.

The barstar gene encodes a barnase-specific inhibitor. It was isolatedfrom Bacillus amyloliquefaciens and is described in Hartley (1998)(Genbank No. X15545).

The plasmid p3222 carries two individually cloned expression cassettes.The first cassette comprises the HMWG promoter, the brittle 2 gene inantisense orientation and the Nos terminator. The second cassettecomprises the A9 promoter, the barstar gene and the CaMV terminator.

The brittle 2 gene was synthesized by PCR from maize albumen cDNA withthe oligonucleotides Bt5 (CCGGATCCATGTGACAGACAGTGTTA, SEQ ID No. 1)containing a BamHI site, and Bt3 (AAGCCCGGGACTTGTACTAACTGTTTC, SEQ IDNo. 2) containing a SmaI site.

The 600 bp PCR fragment thus obtained was digested with SmaI and BamHIand cloned between the HMWG promoter and the nos terminator region ofthe plasmid p3214 opened with SmaI and BamHI. The plasmid p3215 thusobtained contains the HMWG promoter-brittle 2 (antisenseorientation)-nos expression cassette.

A 270 bp fragment containing the barstar gene was amplified by PCR fromthe plasmid pWP127 with the oligonucleotides BPR5(TATCGGATCCAAATCATAAGAAAGGAG, SEQ ID No. 3) containing a BamHI site, andBPR4 (GAAGATCTATATTGTTCATCCCATTG, SEQ ID No. 4) containing a Bg1II site.The PCR fragment thus obtained was digested with BamHI and Bg1II andcloned between the A9 promoter and the CaMV terminator region of theplasmid p1415 opened with BamHI. The plasmid p3072 thus obtainedcontains the barspar genes under the control of the A9 promoter and ofthe CaMV terminator region. The plasmid p3222 according to Example 2.a)corresponds to the insertion of the cassette HMWG promoter-brittle 2(antisense orientation)-nos (KpnI-SacI fragment) of p3215 into p3072opened with KpnI and SacI.

2.b) Construction of a Plasmid (p3223, FIG. 6) Comprising the AntisenseSequence of the Shrunken 2 Gene and also the Fertility-Restoring BarstarGene

The shrunken 2 gene encodes the other subunit of ADP glucosepyrophosphorylase, an enzyme involved in starch synthesis. In theantisense orientation, it makes it possible to inhibit the synthesis ofthis subunit, which produces a mutant phenotype in which the seed sizeis 40% less than the normal size.

The plasmid p3223 carries two individually cloned expression cassettes.The first cassette comprises the HMWG promoter, the shrunken 2 gene inantisense orientation and the Nos terminator. The second cassettecomprises the A9 promoter, the barspar gene and the CaMV terminator.

The shrunken 2 gene was synthesized by PCR from maize albumen cDNA withthe oligonucleotides New Sh5 (GCACCCGGGAGGAGATATGCAGTTTG, SEQ ID No. 5)containing an SmaI site, and Sh3 (GACTGCAGCACAAATGGTCAAG, SEQ ID No. 6)containing a PstI site.

The 1800 bp PCR fragment thus obtained was digested with SmaI and PstIand cloned between the HMWG promoter and the nos terminator region ofthe plasmid p3214 opened with SmaI and PstI. The plasmid p3217 thusobtained contains the HMWG promoter-shrunken 2 (antisenseorientation)-nos expression cassette.

A 270 bp fragment containing the barstar gene was amplified by PCR fromthe plasmid pWP127 with the oligonucleotides BPR5(TATCGGATCCAAATCATAAGAAAGGAG, SEQ ID No. 7), containing a BamHI site,and BPR4 (GAAGATCTATATTGTTCATCCCATTG, SEQ ID No. 8) containing a Bg1IIsite. The PCR fragment thus obtained was digested with BamHI and Bg1IIand cloned between the A9 promoter and the CaMV terminator region of theplasmid p1415 opened with BamHI. The plasmid p3072 thus obtainedpossesses the barstar gene under the control of the A9 promoter and ofthe CaMV terminator region.

The plasmid p3223 according to Example 2.b) corresponds to the insertionof the cassette HMWG promoter-shrunken 2 (antisense orientation)-nos(KpnI-SacI fragment) of p3217 into p3072 opened with KpnI and SacI.

2.c) Construction of a Plasmid (pRec 4962) Comprising the AntisenseSequences of the Shrunken 2 and Brittle 2 Genes and also theFertility-Restoring Barstar Gene

The plasmid p4962 (FIG. 7) carries two individually cloned expressioncassettes, the first cassette comprising the HMWG promoter, the shrunken2 gene in antisense orientation, the brittle 2 gene in antisenseorientation and the Nos terminator and the second cassette comprisingthe Mac2.1 promoter, the barstar gene and the CaMV terminator. Thisplasmid was constructed by means of conventional molecular biologytechniques known to those skilled in the art.

The plasmid p4962 is in the form of a donor vector derived from thevector pSB12 (Japan Tobacco, EP 672 752) of approximately 11.7 kb,comprising:

-   -   an ori region: Col E1 plasmid origin of replication, necessary        for the maintenance and the multiplication of the plasmid in the        bacterium,    -   a spectinomycin-resistance gene which is expressed only in the        bacteria,    -   a T-DNA comprising the male fertility-restoring gene (barstar        gene) and the antisense sequences of the shrunken 2 and brittle        2 genes conferring a “small seed” phenotype.

In the present example, the barstar gene is under the control of theMac2.1 promoter and of the CaMV 3′ terminator, the shrunken 2 andbrittle 2 genes in the antisense orientation being under the control ofthe HMWG promoter and of the Nos 3′ terminator. The superbinary vectorpRec 4962 is obtained by homologous recombination of an acceptor plasmidpSB1 (Japan Tobacco, EP 672 752), derived from an Agrobacteriumtumefaciens Ti plasmid, with the donor plasmid p4962.

Example 3 Selection Plasmid

3.a) Construction of a Plasmid (pDM 302, FIG. 8) Comprising the Bar GeneConferring Resistance to the Herbicide Basta® used as Selection Plasmidin Cotransformation with the Restorer Plasmid

As indicated in Example 1, the bar gene makes it possible to select thetransformed plants which are resistant to the herbicide Basta®.

The plasmid pDM302 (Cao et al., 1992) carries the expression cassettecomprising the actin promoter-actin intron, the bar gene and the Nosterminator.

This plasmid pDM302 was obtained in the following way:

The coding region of the Streptomyces hygroscopicus bar gene encodingPAT (phosphinothricin acetyl transferase) activity was excised from theplasmid pIJ4104 (White et al., 1990) by means of the SmaI restrictionenzyme (600 bp fragment) and cloned into the expression vector pCOR113(McElroy et al., 1991) behind the 5′ fragment (promoter and firstintron) of the rice actin 1 gene (Act-1). This generated the 4.9 kbplasmid pDM301 containing the Act1-bar expression cassette. The Act1-barexpression cassette of pDM301 was excised as a 2.0 kb XhoI-XbaIrestriction fragment and cloned between the SalI and XbaI sites upstreamof the terminator sequence of the nos gene encoding nopaline synthase(plasmid pNOS72). The 4.7 kb pDM302 plasmid thus obtained contains theAct1-bar-nos expression cassette.

3.b) Construction of a Plasmid pRec 273 Comprising the Bar GeneConferring Resistance to the Herbicide Basta® used as a SelectionPlasmid in Cotransformation with the Restorer Plasmid

The plasmid pBIOS 273 (FIG. 9) carries an expression cassette comprisingthe rice actin promoter, the bar gene and the Nos 3′ terminator. Thisplasmid was constructed by means of conventional molecular biologytechniques known to those skilled in the art.

The plasmid pBIOS 273 is in the form of a donor vector derived from thevector pSB12 (Japan Tobacco, EP 672 752), of approximately 8.6 kb,comprising:

-   -   an ori origin: Col E1 plasmid origin of replication, necessary        for the maintenance and the multiplication of the plasmid in the        bacterium,    -   a spectinomycin-resistance gene which is expressed only in the        bacteria,    -   a T-DNA comprising the bar gene conferring resistance to the        herbicide Basta® under the control of the rice actin promoter        and of the Nos 3′ terminator.

The plasmid pBIOS 273 was generated in two steps:

-   -   cloning of the BspDI/XhoI fragment (actin promoter-bar gene-Nos        terminator) of the vector pDM 302 (Cao et al., 1992) into the        SmaI and BspDI sites of the vector pSB12 (Japan Tobacco, EP 672        752). The vector resulting from this cloning is called pBIOS        272.    -   delection of the XhoI site at position 3363 of the vector pBIOS        272 by partial digestion with XhoI and the action of DNA        polymerase I, large (Klenow) fragment. The vector obtained,        which has a unique XhoI site, is called pBIOS 273.

The superbinary vector pRec 273 is obtained by homologous recombinationof an acceptor plasmid pSB1 (Japan Tobacco, EP 672 752), derived from anAgrobacterium tumefaciens Ti plasmid, with the donor plasmid pBIOS 273.

Example 4 Production of a Male Sterile Maize Line Heterozygous for theAMS Transgene

4.a) Production of a Male Sterile Maize Line Heterozygous for the AMSTransgene (AMS/+) by Transformation of Maize with the Plasmid DescribedAccording to Example 1a (pRec 274)

A heterozygous male sterile maize line expressing the barnase(conferring male sterility) and bar (glufosinate resistance) genesrespectively under the control of the A9 (Paul et al., 1992) andactin-intron (McElroy et al., 1991) promoters is obtained bytransformation with Agrobacterium tumefaciens according to the methoddescribed by Ishida et al. (1996).

In the following example, the heterozygous (AMS/+) male sterile maizeline was produced by the Agrobacterium tumefaciens transformationmethod. Other transformation techniques known to those skilled in theart may be used.

Obtaining and Preparation of the Plant Material:

Maize ears are decontaminated for 15 to 20 minutes in 20% Domestos withstirring and are then rinsed with sterile water before removing theimmature embryos, which are placed in LSinf medium. The optimal size ofthe immature embryos is from 1 to 1.2 mm, which corresponds to 10+/−2days after fertilization. The embryos are then vortexed, the LSinfmedium is removed, and rinsing in LSinf medium is carried out beforevortexing again.

Preparation of the Bacteria:

Agrobacterium tumefaciens bacteria (strain LBA 4404) containing thesuperbinary plasmid pRec 274 (as described in Example la) are placed inculture in YP medium supplemented with a selective agent suitable forthe strain. 2 to 3 days later, the bacteria are suspended in LSinfmedium+100 μM acetosyringone. The concentration of the inoculum isconsidered to be at 1×10⁹ bacteria/ml.

Inoculation and Coculturing:

After having removed the LSinf medium, the embryos are brought intocontact with the agrobacteria. After having vortexed, 50 μl of 1%Pluronic F68 are added and incubation is carried out for 5 minutes atambient temperature. The inoculum is removed and the embryos arerecovered and placed on 1.5LSA medium, scutellum facing upwards. Afterhaving sealed the dish, incubation is carried out at 25° C. in the darkfor 5 days.

Selection of Transformed Calluses:

At the end of the coculturing, the embryos are transferred onto LSD5medium and placed in a proportion of 25 per dish sealed with Urgopore.Incubation is carried out at 25° C. in the dark for 2 weeks (1^(st)selection). A 2^(nd) selection consists in transferring the embryos ontoLSD10 medium, by cutting the germinations. Incubation is carried out for3 weeks under the same conditions as in the 1^(st) selection. A 3^(rd)selection is carried out by excising the “good” type I calluses so as toobtain fragments of 1-2 mm. They are placed in culture on LSD10 medium,followed by incubation for 3 weeks under the same conditions as in thefirst and second selections.

Regeneration of Transformed Plantlets:

The type I calluses which have proliferated are placed on LSZ2 mediumand the dishes are sealed with scellofrais® and placed in a culturechamber at 27° C. for 2 weeks. The type I calluses which haveproliferated are again collected, separated and placed on RM+G4C100medium. The dishes are sealed with scellofrais® and placed in a culturechamber at 27° C. The regenerated plantlets are subcultured on T1G4medium and placed under continuous light at 27° C. for one to two weeks.The plantlets which have attained a sufficient development aretransferred to a phytotron.

In order to identify the plantlets resistant to the herbicide Basta®,and which have therefore integrated the transgene, a selection step iscarried out with a Basta F1 solution (AgrEvo France). This solution isapplied by leaf painting on maize plants at the 4- to 5-leaf stage. Theammonium glufosinate concentration in the treating solution is 0.75grams per liter.

The resistant plants exhibit a non-necrosed region 5 days afterapplication of the herbicide. The sensitive plants exhibit necrosis inthe treated region; death of the chlorophyll-containing tissues is thenobserved.

The plants thus regenerated are acclimatized and then cultivated underglass, where they can be crossed or self-fertilized.

4.b) Production of a Male Sterile Maize Line Heterozygous for the AMSTransgene (AMS/+) by Transformation of Maize with the Plasmid DescribedAccording to Example 1b (pRec 424)

A heterozygous male sterile maize line expressing the barnase geneconferring male sterility, under the control of the A9 promoter (Paul etal., 1992), is obtained by transformation with A. tumefaciens accordingto the method described by Ishida et al. (1996). The Agrobacteriumtumefaciens transformation technique, with no implied limitation, usedin this example is identical to that used in Example 4.a.

Transformation with the expression cassette A9-barnase-CamV 3′-Ds::NPTIIcomprising the barnase gene under the control of the A9 promoter and ofthe CaMV 3′ terminator (according to Example lb) on a vector which canbe used in transformation via Agrobacterium and which comprises the“kanamycin” selection marker within the Ds transposable element has theadvantage of eliminating the marker gene, which will no longer be in thetransformed maize line.

In the present example, the plantlets which have integrated thetransgene are identified in the following way:

Regeneration of Transformed Plantlets:

The type I calluses which have proliferated are placed on LSZ2 mediumand the dishes are sealed with scellofrais® and placed in a culturechamber at 27° C. for 2 weeks. The type I calluses which haveproliferated are again collected, separated and placed on RM+G4C100medium. The dishes are sealed with scellofrais® and placed in a culturechamber at 27° C. The regenerated plantlets are subcultured on T1G4medium and placed under continuous light at 27° C. for one to two weeks.The plantlets which have attained a sufficient development aretransferred to a phytotron.

In order to identify the plantlets resistant to kanamycin and which havetherefore integrated the transgene, a selection step (by the cornet droptest) is carried out with a solution of kanamycin at a concentration of500 mg/l, to which 1% of Tween has been added. 2 to 3 drops of thissolution are applied to the maize plants at the 4- to 5-leaf stage.

The plants are analyzed 5 days after application of the kanamycin. Thesensitive plants exhibit the appearance of whitish regions (death of thechlorophyll-containing tissues). The resistant plants do not exhibit theappearance of whitish regions 5 days after application of the kanamycin.

The plants thus regenerated are acclimatized and then cultivated underglass, where they may be crossed or self-fertilized.

Once the transformants (comprising the A9-barnase-CaMV 3′-Ds::NPTIIexpression cassette) have been isolated by the action of the selectionagent and/or by molecular analyses, the gene encoding the selectionagent is eliminated. The selected transformants are therefore crossedwith an active transposase source Ac.

The fertilization is carried out manually using a technique known tothose skilled in the art by depositing the pollen from the transposasesource Ac onto the bristles of the transformants, preferably in thedirection of the male plant possessing the transposase into the femaleplant containing the Ds element::NptII.

The F1 seeds are germinated so as to obtain plantlets. The plantlets areevaluated for their kanamycin resistance (the resistant plants areconserved) and a PCR test is carried out to detect the somatic excisions(a somatic excision does not generally affect the gametes and results inthe formation of chimeric seeds and individuals in which most of thecells still possess the gene encoding the selection agent). The primersused for this PCR test which makes it possible to search for somaticexcisions on the F1 plants resistant to the selection agent are asfollows: Name Size (bp) Sequence Barn5 21 5′-GGTTTCGCTCATGTGTTGAGC-3′(SEQ ID No. 15) EM11 25 5′-CATTGCGGACGTTTTTAATGTACTG-3′ (SEQ ID No. 16)

The pair of primers Barn5/EM11 makes it possible to visualize theexcision (other appropriate primers can also be used). The amplificationis different depending on whether or not excision has taken place. TheF1 plants in which somatic excision has taken place are thereforeselected and then crossed with a plant having a wild-type genotype (WT).The F2 plants are screened in order to identify the plants with the geneof interest without the gene encoding the selection agent (a germinalexcision affects the gametes and results in the formation of seeds andof individuals consisting of cells which no longer possess the geneencoding the selection agent).

4.c) Molecular Characterization of the Transformants

The Southern methodology (1975) is used to demonstrate the insertion ofthe transgene into the genome of the plant and to evaluate the number ofcopies and to characterize the integration profile.

The genomic DNA of the plants (8 μg) is extracted from the leaves of theplants according to a CTAB (cetyltrimethylammonium bromide) extractionprotocol, according to the protocol of J. Keller (DNAP 6701 San PabloAve Oakland Calif. 94608 USA) modified by I. Bancroft (Department ofMolecular Genetics, Cambridge Laboratory, John Innes Center for PlantScience Research, Colney lane, Norwich, England). The DNA obtained wasdigested with various restriction enzymes, separated by agarose gelelectrophoresis and transferred onto hybond N+nylon membrane and thenhybridized with radioactive probes.

The Bar probe for the molecular analyses is prepared in the followingway: the plasmid pDM302 (described in Example 3a) of the presentinvention) is digested with the SmaI enzyme. The 0.6 kb fragment ofinterest is recovered after migration of the digestion product onelectrophoresis gel and purification with the Gene Clean kit (Bio 101,Ozyme). After P32-labeling of 30 ng of fragment, said fragment is usedas probe for hybridizing the various blots.

The male sterile transformation event (STB27b) is a transformantobtained by transformation via Agrobacterium tumefaciens according toExample 4a) described above, and which has a single-copy insertion ofthe T-DNA as described according to Example la. The results fromhybridization of the DNA digested with various restriction enzymes(NcoI, SpeI, EcoR V, HindIII and Ecor I), and then hybridized with theBar probe, show that a single fragment is revealed whatever the enzymeused. The size of the fragment revealed by the HindIII digestion is 1.7kb. This same type of molecular characterization is carried out for thetransformants obtained by transformation by Agrobacterium tumefaciensaccording to Example 4b).

Example 5 Production of a Fertility-Restoring Maize Line Heterozygousfor the Fertility-Restoring Gene (SSB/+)

5.a) Production of a Fertility-Restoring Maize Line (SSB/+) Heterozygousfor the Fertility-Restoring Gene by Cotransformation of Maize with thePlasmids of Examples 2a, 2b (Restorer Plasmids) and 3a) (SelectionPlasmid pDM 302)

A method of genetic transformation which results in the stableintegration of the modified genes into the plant's genome is preferablyused. This method is based on the use of a particle gun. However, othertransformation techniques known to those skilled in the art may be used.

Production of Immature Embryos:

It consists in the self-fertilization of a plant of the HiII line or ofthe “brother-sister” (sib) crossing of 2 plants of the HiII line. Thefertilization is carried out on a single date after having isolated thereproductive organs.

Ear Removal:

The ears are removed when the immature embryos have reached a size of1.5 mm to 2 mm, i.e. 10 to 11 days after fertilization under ourcultivation conditions (temperature of 25° C. during the day and 18° C.at night, 16/8 photoperiod).

Disinfection:

The harvested ears have their spathes and their bristles removed and arethen disinfected with 20% (v/v) Domestose for 15 minutes with stirring.The ears are rinsed three times with sterile water.

Embryo Extraction:

The upper part of the seed is cut so as to reveal the albumen, and thena slight pressure on the seed makes it possible to free the albumen. Theimmature embryo which is still in the seed (against the pericarp) isextracted and then placed on the callogenesis medium N6P6, flat sideplaced on the agar. Thirty-six embryos per dish are placed in culturefor 4 days in a culture chamber at 26° C. and in the dark. This is theinitiating period.

Genetic Transformation:

Preparation of the Embryos:

After the initiating period, the embryos are placed, 4 hours before thefiring, on the 0.4 M N6P6 osmotic shock medium and are arranged in aproportion of 36 in a small square 2 cm² at the center of the dish. Thedishes are sealed with scellofrais and incubated in a culture chamber(26° C. in the dark).

The plasmids carrying the genes to be introduced (plasmids described inExamples 2a, 2b and 3a) are purified on a Qiagen® column according tothe manufacturer's instructions. They are then precipitated ontoparticles of tungsten (M10) according to the protocol described by Klein(1987). The particles thus coated are projected onto the target cellsusing a gun and according to the protocol described by J. Finer (1992).

The dishes of embryos thus bombarded are then sealed with scellofrais®and cultivated in the dark at 27° C. The first subcultivation takesplace 24 h later, and then every fifteen days for 3 months on mediumidentical to the initiating medium, supplemented with a selective agent,the nature and the concentration of which may vary according to the geneused. The selective agents which can be used generally consist of activecompounds of certain herbicides (Basta®, Round up®) or certainantibiotics (hygromycin, kanamycin, etc.). Preferably, the gene forresistance to the herbicide Basta® will be used.

Maturation and Regeneration of Type II Calluses:

When there is sufficient material for an event, it is transferred ontothe MM+G2 maturation medium which promotes the development of somaticembryos. The callus is plated out at the surface of the MM+G2 medium.The dishes are placed in a culture chamber at 26° C. in the dark. After15 days (minimum) on the MM+G2 medium, somatic embryos appear. Thelatter are subcultivated on RM+G2 regeneration medium (20 to 25 perdish) and grown at 28° C. under light (16 h/24 h). It is considered that4 regeneration dishes are sufficient to obtain regenerants.

After approximately 15 days on the RM+G2 medium, the embryos havedeveloped into plantlets which are then subcultivated in tubes on theT1+G2 rooting medium. It is considered that 5 plantlets per event arenecessary to achieve the acclimatization in a phytotron and thestart-ups. These plantlets are placed in a culture chamber under light.

After 3 months, or sometimes earlier, calluses are obtained, the growthof which is not inhibited by the selection agent (ammonium glufosinate),and which are usually and mainly made up of cells resulting from thedivision of a cell having integrated into its genetic inheritance one ormore copies of the selection gene. The transformation efficiency is 10%.

These calluses are identified, individualized, amplified and thencultivated so as to regenerate plantlets. In order to avoid anyinterference with nontransformed cells, all these operations are carriedout on culture media containing the selective agent.

Acclimatization:

The acclimatization of the plantlets is carried out when the latter havedeveloped sufficiently on T1+G2, i.e. when the roots reach the bottom ofthe tube and when the ridged axis is sufficiently rigid and developed.The plantlets are acclimatized in a phytotron in small pots withslightly enriched compost. The small pots are arranged on a benchlocated 1.5 meters from the lamps. To maximize the obtaining of progeny,the acclimatization of 2 plantlets in a phytotron is necessary. Onaverage, two weeks are necessary for weaning the plantlets.

The plants thus regenerated and acclimatized are then cultivated in agreenhouse where they can be crossed or self-fertilized.

This embodiment consisting in producing a fertility-restoring maize lineheterozygous for the fertility-restoring gene (SSB/+) bycotransformation of a maize plant with the plasmids of Examples 2a, 2band 3a is the first embodiment.

A second embodiment consisting in producing a fertility-restoring maizeline heterozygous for the fertility-restoring gene (SSB/+) consists incotransforming a maize plant with the plasmids of Examples 2a and 3aaccording to the same protocol as described above.

A third embodiment consisting in producing a fertility-restoring maizeline heterozygous for the fertility-restoring gene (SSB/+) consists incotransforming a maize plant with the plasmids of Examples 2b and 3aaccording to the same protocol as described above.

5.b) Production of a Fertility-Restoring Maize Line Heterozygous for theFertility-Restoring Gene (SSB/+) by Cotransformation of Maize with thePlasmids of Examples 2c (Restorer Plasmid pRec 4962) and 3b (SelectionPlasmid pRec 273)

The production of the fertility-restoring maize line according to thepresent Example 5b is carried out by cotransformation of a maize plantwith the plasmids described according to Examples 2c and 3b according tothe Agrobacterium tumefaciens transformation technique known to thoseskilled in the art.

The bar gene (gene encoding the selection agent) is eliminated duringthe cotransformation. 10% of the transformants obtained contain the 2expression cassettes contained in the plasmids of Examples 2c and 3b.There will be segregation in the progeny.

This embodiment (4^(th) embodiment) consisting in producing afertility-restoring maize line heterozygous for the fertility-restoringgene (SSB/+) is the preferred embodiment according to this presentinvention. However, other transformation techniques known to thoseskilled in the art may be used.

5.c) Molecular Characterization of the Transformants

The transformant SSB-001a obtained according to Example 5a) wascharacterized by the same methodology as that described according toExample 4c of the present invention. The molecular analysis was carriedout on the transformant SSB-001a resistant to the herbicide Basta®(plant No. 12928), on a sister plant resistant to the herbicide Basta®(plant No. 12929) and on 2 plants sensitive to the herbicide (13008 and13061). These 4 plants are derived from the ear of the primarytransformant SSB-001a. The genomic DNA (14 μg) of the plants wasdigested with the Eco RV and Hind III restriction enzymes. Three probeswere used for the analysis: A9 promoter (pA9), HMWG promoter (pHMWG)and. Bar.

The results of the molecular hybridizations with the Bar probe indicatethat the molecular profiles are identical for the two plants resistantto the herbicide Basta®. The hybridizations with the pA9 and pHMWGprobes reveal a large number of bands, reflecting the integration ofseveral copies of the plasmids p3222 and p3223. The hybridization withthe Bar probe shows a simple profile, a major band is revealed for the 2enzymes Eco RV and Hind III. The size of the fragment revealed by theHind III digestion is 8 kb. The bands of very weak intensity correspondto the very intense signals revealed previously with the pA9 and pHMWGprobes.

Example 6 Crossing of the Heterozygous Male Sterile Maize Line (AMS/+)with the Fertility-Restoring Maize Line Heterozygous for theFertility-Restoring Gene (SSB/+): Obtaining F1 Plants

The heterozygous male sterile maize line (AMS/+) resistant to theherbicide Basta® obtained according to Example 4a is crossed with thefertility-restoring maize line heterozygous for the fertility-restoringgene (SSB/+), plant resistant to the herbicide Basta®, obtainedaccording to Example 5 in order to obtain the F1 plants. The same typeof cross can be carried out between the heterozygous male sterile maizeline (AMS/+) obtained according to Example 4b and thefertility-restoring maize line heterozygous for the fertility-restoringgene (SSB/+), obtained according to Example 5. The fertilization iscarried out manually by means of a technique known to those skilled inthe art. The male sterile plant is brought to flowering, and the pollenof the (SSB/+) plant is deposited onto the bristles of the male sterileline.

At the end of this cross, genetic analyses are carried out on the F1plants. The genetic analyses consist of counting, in relation to thepresence of the various markers, among the progeny.

All the theoretical frequencies mentioned are true if, and only if,there is no linkage between the AMS transgene and thefertility-restoring gene.

Genetic Assessment of the F1: AMS + SSB SSB/+; AMS/+ SSB/+; +/+ + AMS/+;+/+ +/+; +/+

Phenotypic Assessment of the F1: Seed Seed Basta segregation phenotypesegregation Resistant Sensitive Normal 50% 50% 50% Deficient 50% 100% 0%

Half the F1 is therefore made up of normal seeds, the other half beingdeficient seeds (“small seed” phenotype caused by the shrunken2 andbrittle2 genes in the antisense orientation).

For each F1, we evaluated the segregation for the glufosinate resistancein order to detect the batches of deficient seeds.

The deficient seeds are selected by visual separation with respect tothe maize ear. These seeds are all resistant to the herbicide Basta®.

The deficient seeds, which have the genotype (AMS/+; SSB/+) or (+/+;SSB/+), are selected and then sown and germinated.

Example 7 Self-Fertilization of the F1 Plants for Production of the F2

The F1 plants resistant to the herbicide Basta® derived from deficientseeds according to Example 6 are self-fertilized in order to obtain theF2 plants.

Since the F1 plants derived from deficient seeds consist of both plantsof genotype (SSB/+; +/+) and plants of genotype (SSB/+; AMS/+), thereare then two cases of self-fertilization:

7.a) Self-Fertilizations of the Plants of Genotype (SSB/+; +/+)

The F1 plants of genotype (SSB/+; +/+) obtained according to Example 6are self-fertilized. At the end of this self-fertilization, the geneticanalyses are carried out on the F2 plants:

Genetic Assessment of the F2: SSB + SSB SSB/SSB; +/+ SSB/+; +/+ + SSB/+;+/+ +/+; +/+

Phenotypic Assessment of the F2: Seed Seed Basta segregation phenotypesegregation Resistant Sensitive Normal 25% 0% 100% Deficient 75% 100% 0%

This step comprising self-fertilizations of the plants of genotype(SSB/+; +/+) can advantageously be eliminated by means of a stepconsisting in genotyping by carrying out a PCR specific for the AMStransgene on the F1 plants derived from deficient seeds according toExample 6. The deficient seeds are germinated, the young maize plantsare self-fertilized, and molecular separation is carried out by PCR.Only the plants positive for detection of the AMS transgene by PCR areconserved.

The primers specific for the AMS gene of interest (pA9-barnase-CaMV3′)which allows its amplification by PCR are as follows: Name OrientationSequence A9A Direct TAGACATTGTAGGTTGGTTTTG (SEQ ID No. 9) (Pro A9) Barn1 Direct GCACAGGTTATCAACACGTTTGAC (SEQ ID No. 10) (barnase) Barn 4Direct ATCCGGCCATTTCTGAAGAGAA (SEQ ID No. 11) (barnase) A9B ReverseTCTAGTTACTTCATAAGTTTTG (SEQ ID No. 12) (Pro A9) Barn 6 ReverseTTGCGGGTTTGTGTTTCCATATTG (SEQ ID No. 13) (barnase) CaMVol1 ReverseATTGATAAGGGGTTATTAGGGG (SEQ ID No. 14) (3′ CaMV)

The size of the fragment amplified using the pairs of primers A9A/A9B,Barn1/Barn6 or Barn4/CaMVoll is 799 bp, 880 bp or 979 bp, respectively.

7.b) Self-Fertilizations of the Plants of Genotype (AMS/+; SSB/+)

The F1 plants of genotype (AMS/+; SSB/+) obtained according to Example 6are self-fertilized in order to obtain the F2 progeny. At the end ofthis self-fertilization, the genetic analyses are carried out on the F2progeny:

Genetic Assessment of the F2: AMS; + +; SSB AMS; SSB +; + AMS; +AMS/AMS; AMS/+; AMS/AMS; AMS/+; +/+ SSB/+ SSB/+ +/+ +; SSB AMS/+; SSB/+SSB/SSB; AMS/+; SSB/SSB SSB/+; +/+ +/+ AMS; AMS/AMS; AMS/+; AMS/AMS;AMS/+; SSB SSB/+ SSB/SSB SSB/SSB SSB/+ +; + AMS/+; +/+ SSB/+; +/+ AMS/+;SSB/+ +/+; +/+

Phenotypic Assessment of the F2: Seed Seed Basta segregation phenotypesegregation Resistant Sensitive Normal 25% 75% 25% Deficient 75% 100% 0%

The genetic analysis of the F2 progeny on the seeds that are normal(25%) for the expression of the barnase gene with resistance to theherbicide Basta® makes it possible to determine the F1 hybrid plantswhich exhibited the genotype (SSB/+; AMS/+). In terms of the F2 plantsderived from the deficient seeds, ⅙ have the genotype (AMS/AMS; SSB/+).As regards the F2 plants which have normal seeds, ¼ have the genotype(AMS/AMS; +/+).

7.c) Results

14 F2 ears were produced. For each of these ears, separation was carriedout with respect to seed genotype (deficient or not). 14 batches oftransgenic seeds (from A to N) were therefore analyzed. These 14 seedbatches (batch A to batch N) were divided in two as a function of theirnormal or deficient phenotype. The code used for the 28 sets of seedsthus prepared is, for example, for batch A: A01=normal seeds,A02=“deficient” seeds.

Example 8 Sowing of the Deficient Seeds of the F2 Progeny and Genotypingof the Plants (AMS/AMS; SSB/+)

Directed sowing of the F2 seeds obtained above, as a function of thedeficient or normal seed phenotype, and then detection of the (AMS/AMS;SSB/+) plants by genotyping were carried out.

8.a) Sowing of the F2 Progeny

Directed sowing of the 14 families was carried out and, at the 3- to4-leaf stage, a 0.5% Basta spray test was carried out. The results ofthis test are described in the table below: Number of Number of Numberof Number of germinations resistants sensitives Seed seeds / seeds /seeds / seeds Code phenotype sown sown % sown % sown % A 01 normal 30 30100 27 90 3 10 A 02 deficient 60 60 100 60 100 0 0 B 01 normal 30 2893.3 0 0 24 85.7 B 02 deficient 60 60 100 60 100 0 0 C 01 normal 30 30100 19 63.3 11 36.7 C 02 deficient 60 59 98.3 56 94.9 3 5.1 D 01 normal30 30 100 20 66.7 10 33.3 D 02 deficient 60 60 100 57 95 3 5 E 01 normal30 30 100 19 63.3 11 36.7 E 02 deficient 50 48 96 48 100 0 0 F 01 normal30 30 100 2 6.7 28 93.3 F 02 deficient 60 60 100 60 100 0 0 G 01 normal30 29 96.7 21 72.4 8 27.6 G 02 deficient 60 59 98.3 59 100 0 0 H 01normal 15 15 100 7 46.7 8 53.3 H 02 deficient 30 29 96.7 29 100 0 0 I 01normal 30 30 100 0 0 24 80 I 02 deficient 60 60 100 60 100 0 0 J 01normal 30 30 100 0 0 27 90 J 02 deficient 60 59 98.3 55 93.2 4 6.8 K 01normal 30 30 100 21 70 9 30 K 02 deficient 60 60 100 58 96.7 5 8.3 L 01normal 30 30 100 0 0 29 96.7 L 02 deficient 60 60 100 60 100 0 0 M 01normal 30 30 100 22 73.3 7 23.3 M 02 deficient 60 60 100 60 100 0 0 N 01normal 30 28 93.3 20 71.4 7 25 N 02 deficient 60 60 100 60 100 0 0

The results of the test made it possible to eliminate the families B, F,I, J and L which resulted from the self-fertilization of F1 plants withthe genotype (SSB/+; +/+).

The families A, C, D, E, G, H, K, M and N were conserved. These earsresulted from the self-fertilization of F1 plants with the genotype(SSB/+; AMS/+). Details of the genetic and phenotypic assessments of theself-fertilization of these plants were given in Example 7b. In theseselected families, all the plants resistant to the herbicide Basta®derived from normal seeds (batches ending with 01) and up to 40 plantsresistant to the herbicide Basta® derived from deficient seeds were keptfor cultivation.

8.b) Identification of the Plant Genotype (AMS/AMS; SSB/+) by Genotypingand Southern Blotting

A molecular analysis is carried out in order to identify the plantshomozygous to the transgene derived from STB-27b (AMS/AMS) andheterozygous for the transgene derived from SSB001a (SSB/+).

The genomic DNA of the progeny was extracted from 50 mg of leavesaccording to the protocol and use of the extraction kit: Qiagen Dneasy96 plant kit (Qiagen SA, 91974 Courtaboeuf cedex, France). Southernmethodology is used to identify the molecular profiles.

For each individual, the presence of the two Bar genes: that coming fromSTB27b and that coming from SSB001a, is visualized. For this, the DNAswere digested with the Hind III enzyme and then hybridized with the Barprobe. According to the molecular analyses carried out on the parenttransformants, it is known that a fragment 8 kb in size corresponds tothe copy of the Bar gene derived from the transformant SSB001a and afragment 1.7 kb in size corresponds to the copy of the Bar gene derivedfrom the transformant STB27b. For a given plant, the intensity of thehybridization signals is also evaluated, in order to identify thezygosity (heterozygosity or homozygosity) of the plants for the Bar geneunder consideration (SSB001a or STB27b). This analysis therefore allowsus to identify the genotype of the plants derived from the SSB×STBcross. According to the prior selection of the seeds from which theplants are derived, 6 genotypes are expected and presented in Table 1.Table 1: Genotype Number of copies of Number of copies of Bar fromSSB-001a Bar from STB-27b theoretical % 2 0 A 8.3 2 1 B 16.7 2 2 C 8.3 10 D 16.7 1 1 E 33.3 1 2 F 16.7 Total 100

The genotype sought in this study is homozygosity for the STB-27b T-DNA(“2 copies” of the Bar gene) and heterozygosity for the SSB-001a Bargene (“1 copy” of the Bar gene).

Southern blots were performed with the DNA from 8 daughter plantsderived from an ear, on the 9 ears selected. 58 plants were genotypedaccording to the protocol described above. All the expected genotypesare represented among the 9 plants analyzed (2 different ears).

In total, 10 plants exhibit the genotype of interest, namely:homozygosity for the STB-27b T-DNA (“2 copies” of the Bar gene) andheterozygosity for the SSB-001a Bar gene (“1 copy” of the Bar gene). Thefrequencies observed are very close to the expected theoreticalfrequencies whatever the genotype under consideration (Table 2). TABLE 2Genotype Number of Number of Number copies of Bar copies of Bar of fromSSB-001a from STB-27b plants observed % theoretical % 2 0 3 5.2 8.3 2 19 15.5 16.7 2 2 3 5.2 8.3 1 0 11 19 16.7 1 1 22 37.9 33.3 1 2 10 17.216.7 Total 58 100 100

Example 9 Obtaining Prebase Seeds (AMS/AMS; +/+), Base Seeds (AMS/+;+/+) and Hybrid Seeds

Many crosses can be envisioned in order to produce prebase seeds, baseseeds and hybrid seeds. The crosses described below are are notlimiting:

9.a) Self-Fertilization of the Plants (AMS/AMS; SSB/+) in Order toObtain Prebase Seeds

The advantage of this cross is to multiply the line with the genotype(AMS/AMS; SSB/+) and also to produce seeds with the genotype (AMS/AMS;+/+) or prebase seeds.

In fact, self-fertilizations produce seed homozygous for the transgeneconferring male sterility (at the F3 level, all the normal seeds willhave the genotype (AMS/AMS; +/+)). At the end of thisself-fertilization, the genetic analyses are carried out:

Genetic Assessment: AMS; SSB AMS; + AMS; SSB AMS; + AMS; AMS/AMS;AMS/AMS; AMS/AMS; AMS/AMS; SSB SSB/SSB SSB/+ SSB/SSB SSB/+ AMS; +AMS/AMS; SSB/+ AMS/AMS; AMS/AMS; AMS/AMS; +/+ SSB/+ +/+ AMS; AMS/AMS;AMS/AMS; AMS/AMS; AMS/AMS; SSB SSB/SSB SSB/+ SSB/SSB SSB/+ AMS; +AMS/AMS; SSB/+ AMS/AMS; AMS/AMS; AMS/AMS; +/+ SSB/+ +/+

Phenotypic Assessment: Seed Seed Basta segregation phenotype segregationResistant Sensitive Normal 25% 100% 0% Deficient 75% 100% 0%

All of the normal seeds have the genotype (AMS/AMS; +/+). These are theprebase seeds. 75% of the deficient seeds have these genotype (AMS/AMS;SSB/+). The plants derived from these deficient seeds can beself-fertilized in order to multiply (maintain) the plants of genotype(AMS/AMS; SSB/+). The young plant of genotype (AMS/AMS; SSB/+) is alsocalled sterility-maintaining young plant.

9.b) Crossing of the Plants of Genotype (AMS/AMS; SSB/+) with the EliteLine Having a Wild-Type Genotype (WT) in Order to Obtain Seeds ofGenotype (AMS/+; +/+)

The plants derived from deficient seeds are crossed with a WT eliteline. At the end of this cross, the genetic analyses are carried out:

Genetic Assessment: AMS; SSB AMS; + AMS; SSB AMS; + +; + AMS/+; SSB/+AMS/+; +/+ AMS/+; SSB/+ AMS/+; +/+

Phenotypic Assessment: Seed Seed Basta segregation phenotype segregationResistant Sensitive Normal 50% 100% 0% Deficient 50% 100% 0%

Two scenarios occur:

-   -   in ⅔ of the cases, the F2 plants had the genotype (AMS/+; +/+).        On these F3 ears, only normal seeds with a 50/50 segregation        with respect to the herbicide Basta® are obtained. These ears        are of no interest to us;    -   in ⅓ of the cases, the F2 plants had the genotype (AMS/AMS;        +/+). On these F3 ears, only normal seeds with a genotype        (AMS/+; +/+) 100% resistant to the herbicide Basta® are        obtained. These are the ears which interest us. The normal seeds        of genotype (AMS/+; +/+) constitute the base seeds.

20 seeds from each F2 ear are sown and the ears of interest areidentified as a function of resistance to the herbicide Basta®.

9.c) Crossing of the Plants of Genotype (AMS/AMS; +/+) with the EliteLine Having a Wild-Type Genotype (WT) in Order to Obtain Seeds ofGenotype (AMS/+; +/+)

The plants derived from the seeds of genotype (AMS/AMS; +/+) are crossedwith an elite line. Preferably, this elite line is identical to thatused in the step of successive backcrosses in order to introgress thegenotype (AMS/AMS; SSB/+).

The interest of this cross is to produce the base seed of genotype(AMS/+; +/+).

Genetic Assessment: AMS AMS + AMS/+; +/+ AMS/+; +/+ + AMS/+; +/+ AMS/+;+/+

Phenotypic Assessment Seed Seed Basta segregation phenotype segregationResistant Sensitive Normal 100% 100% 0%

All the seeds (which are normal) have the genotype (AMS/+; +/+) and,consequently, will give male sterile plants.

9.d) Crossing of the Plants of Genotype (AMS/+; +/+) with the Male EliteLine (WT) in Order to Obtain Hybrid Seeds

Plants derived from the seeds of genotype (AMS/+; +/+) are crossed witha male elite line. Preferably, the elite line used in this cross isdifferent from that used in the cross described in Example 9.c).

The interest of this cross is to produce the hybrid seed.

Genetic Assessment: AMS + + AMS/+; +/+ +/+; +/+ + AMS/+; +/+ +/+; +/+

Phenotypic Assessment: Seed Seed Basta segregation phenotype segregationResistant Sensitive Normal 100% 50% 50%

All the seeds are normal. Half of them will give male sterile plants,the other half will give male fertile plants, thus ensuring pollinationand, consequently, the production of maize “for consumption” or “seed”maize.

Example 10 Separation of the Deficient Seeds by Means of a DensimetricTable

The densimetric table used makes it possible to divide up into sixfractions seeds of equivalent size and superficial quality, but whichdiffer from one another by virtue of their specific weight.

Densimetric separation is carried out on a batch of seeds from F3 ears,derived from the self-fertilization of plants of genotype (+/+; SSB/+).Theoretically, these ears comprise 75% of deficient seeds and 25% ofnormal seeds. 33 ears were shelled so as to obtain approximately 500grams of seed. 6 seed fractions were constituted by densimetricseparation.

The table below is a summary of the results obtained (separation withrespect to the phenotype of the seed and genetic analyses regarding theexpression of the bar gene conferring glufosinate resistance). Seedphenotype Genetic analysis results Total seperation Number Number Numbernumber Number of Number of Number of germinations resistants sensitivesof seeds normal seeds deficient seeds seeds sown /seeds sown %/germinations % /germinations % Fraction 1 50 0 50 40 36 90 36 100 0 0Fraction 2 69 0 69 60 49 81.7 49 100 0 0 Fraction 3 445 27 418 160 15697.5 149 96 7 4 Fraction 4 97 8 89 80 80 100 72 90 8 10 Fraction 5 540103 437 160 154 96.3 120 78 34 22 Fraction 6 779 756 23 160 155 96.9 6 4149 96Separation with Respect to the Seed Phenotype (Normal or Deficient):

Fractions 1 and 2 comprise only deficient seeds.

In fractions 3, 4 and 5, the seeds are essentially deficient seeds, butsome normal seeds are also found (i.e. 6.1, 8.2 and 19.1%, respectively,for fractions 3, 4 and 5).

As regards fraction 6, there are virtually only normal seeds (97%).

Fractions 1 to 5 therefore correspond to the deficient seeds andfraction 6 to the normal seeds.

A small quantity of nonseparated seeds is also found.

This small quantity corresponds both to normal seeds and to deficientseeds. However, visually, there is a greater number of deficient seedscompared to the normal seeds.

Genetic Analysis Results (Regarding Expression of the Bar GeneConferring Resistance to the Herbicide Basta®):

The genetic analysis results reinforce those of the separation carriedout with regard to the seed phenotype for each of the fractions.

In fraction 6, there is therefore a small portion of deficient seeds(approximately 3 to 4%). The latter is completely removed through beingpassed over the densimetric table a second time.

Fractions 1 to 5 correspond to the deficient seeds and fraction 6 to thenormal seeds.

Example 11 Evaluation of the Various Steps of the Production of HybridMaize Seeds Using this Novel Method

Three types of experiment were tested for seed production:

-   -   Production of prebase seeds    -   Production of base seeds    -   Product of hybrid seeds        Production of Prebase Seeds:

The production of prebase seeds (genotype (AMS/AMS; +/+)) was providedby the cultivation of plants of genotype (AMS/AMS; SSB/+). These plantsare indeed observed to be male fertile.

The plants of genotype (AMS/AMS; SSB/+) are self-fertilized.Approximately 25% of seeds obtained in the progeny correspond to prebaseseeds.

Production of Base Seeds:

The production of base seeds (genotype (AMS/+; +/+)) was provided by thecultivation of plants of genotype (AMS/AMS; +/+). These plants areindeed observed to be male sterile and exhibit no deleterious effect onthe vegetative aspect of the plant, demonstrating that the use of an AMStransgene in the homozygous state is compatible with the system forproducing hybrid maize seeds as described in the present invention.

The young plants of genotype (AMS/AMS; +/+) were crossed with a youngplant having a wild-type genotype. 100% of the seeds obtained in theprogeny correspond to base seeds.

Production of Hybrid Seeds:

The production of hybrid seeds was obtained by crossing between malesterile plants of genotype (AMS/+; +/+) derived from the base seeds,with a wild-type elite line. All the young plants of genotype (AMS/+;+/+) indeed showed complete male sterility (100% sterility).

Example 12 Production of Hybrid Seeds

Two types of crosses are preferably carried out in seed production.These two crosses make it possible to produce hybrid seed.

12.a) Crossing of the Young Plants of Genotype (AMS/AMS; SSB/+) withYoung Plants of Genotype (AMS/AMS; +/+)

Genetic Assessment: AMS; SSB AMS; + AMS; SSB AMS; + AMS; + AMS/AMS;SSB/+ AMS/AMS; AMS/AMS; AMS/AMS; +/+ SSB/+ +/+ AMS; + AMS/AMS; SSB/+AMS/AMS; AMS/AMS; AMS/AMS; +/+ SSB/+ +/+

Phenotypic Assessment: Seed Seed Basta segregation phenotype segregationResistant Sensitive Normal 50% 100% 0% Deficient 50% 100% 0%

50% of the seeds obtained are normal and all have the genotype (AMS/AMS;+/+). These seeds can be separated by densimetric separation, preferablyusing a densimetric table.

12.b) Crossing of the Plants of Genotype (AMS/AMS; SSB/+) with an EliteLine Having a Wild-Type Genotype (WT)

Genetic Assessment: AMS; SSB AMS; + AMS; SSB AMS; + +; + AMS/+; SSB/+AMS/+; +/+ AMS/+; SSB/+ AMS/+; +/+

Phenotypic Assessment: Seed Seed Basta segregation phenotype segregationResistant Sensitive Normal 50% 100% 0% Deficient 50% 100% 0%

All the normal seeds have the genotype (AMS/+; +/+). These seeds can beseparated by densimetric separation, preferably using a densimetrictable.

This cross makes it possible to gain time in obtaining the hybrid seeds(6 to 12 months less time) compared with the cross described in Example12.a). On the other hand, it requires a surface for production of themaintaining line (of genotype AMS/AMS; SSB/+) which is greater in sizefor an equivalent number of (hybrid) seed hectares.

BIBLIOGRAPHIC REFERENCES

-   -   An et al. (1986) Plant Physiol. 81, 86-91.    -   Anderson, O. D., Green, F. C., Yip, R. E., Halford, N. G.,        Shewry, P. R. and Malpica-Romero, J. M. (1989) Nucl. Acid. Res.        17, 461.    -   Bevan et al. (1984) Nucleic Acid Research, 11, 369-385.    -   Cao J., Duan, X., McElroy, D. and Wu, R. (1992) Plant. Cell.        Rep. 11, 586-591.    -   Cheng W. H., Taliercio E. W. and Chourey P. S. (1996). The        Miniaturel Seed Locus of Maize Encodes a Cell Wall Invertase        Required for Normal Development of Endosperm and Maternal Cells        in the Pedicel. Plant Cell., 8(6):971-983.    -   Chourey, P. S., Nelson, O. E. (1976). The enzymatic deficiency        conditioned by the shrunken-1 mutations in maize. Biochem Genet,        14(11-12):1041-55.    -   Depicker et al., (1982) J. Mol. Appl. Genet., 1, 561-573.    -   Di Fonzo N, et al. (1988) Mol Gen Genet 212(3), 481-7.    -   Finer J. (1992) Plant Cell Report 11: 323-328.    -   Franck et al. (1980) Cell, 21, 285-294.    -   Fromm et al. (1986) Nature, 319, 791-793.    -   Hartley, R.W (1988) J. Mol. Biol. 202, 913-915.    -   Herrera-Estrella et al. (1983) EMBO J. 2, 987-995.    -   Ishida Y, Saito H, Ohta S, Hiei Y, Komari T, Kumashiro T (1996)        Nat. Biotechnol. 14(6):745-50.    -   Jouanin et al. (1987) Plant Sci., 53, 53-63.    -   Klein (1987) Nature 327:70-73.    -   Komari T., Hiei Y., Saito Y., Murai N., Kumashiro T. (1996).        Vectors carrying two separate T-DANs for co-transformation of        higher plants mediated by Agrobacterium tumefaciens and        segregation of transformants free from selection markers. Plant        Journal, 10, 165-174.    -   Krens et al. (1982) Nature, 296, 72-74.    -   Lyzrik L. and Hodges T. (1997). FLP mediated recombination of        FRT sites in the maize genome. Nucleic Acids Research, 44,        123-132.    -   McElroy D., Blowers, A. D., Jenes, B. and Wu, R. (1991) Mol.        Gen. Genet. 231, 150-160.    -   Messing J. et al. (1983) Methods in Enzymology, 101, 20-78.    -   Paul, W., et al. (1992) Plant Mol. Biol. 19: 611-622.    -   Robert et al. (1989) Plant Cell, 1: 569-578.    -   Sambrook, Fritsch & Maniatis, Molecular Cloning: A Laboratory        Manual, Second Edition (1989) Cold Spring Harbor Laboratory        Press, Cold Spring Harbor, New York.    -   Southern E. M. (1975) J. Mol Biol. 98: 503-517.    -   Watson et al. (1994) Ed. De Boeck University, pp. 273-292.    -   White, J., Chang S-YP., Bibb, M. J. and Bibb, M. J. (1990) Nucl.        Acid Res. 18, 1062.    -   Yoder et al. (1993) Bio/Technology, 12, 263-292.

1. A method for the production of maize seeds homozygous for a transgeneconferring artificial nuclear male sterility (“AMS”) and heterozygousfor a fertility-restoring gene linked to a “small seed” phenotypemarker, comprising the steps consisting in: a) crossing a male sterilemaize plant heterozygous for the AMS transgene with afertility-restoring maize plant comprising in its genome afertility-restoring gene linked to a “small seed” phenotype marker, b)selecting, by means of the “small seed” phenotype, the maize seedscomprising in their genome a fertility-restoring gene linked to a “smallseed” phenotype marker, c) self-fertilizing the maize plants derivedfrom seeds selected according to step b), d) selecting the seedshomozygous for the AMS transgene and heterozygous for thefertility-restoring gene linked to a “small seed” phenotype marker.
 2. Amethod for the production of maize seeds homozygous for a transgeneconferring artificial nuclear male sterility (“AMS”) and heterozygousfor a fertility-restoring gene linked to a “small seed” phenotypemarker, comprising the steps consisting in: a) crossing a male sterilemaize plant heterozygous for the AMS transgene with afertility-restoring maize plant comprising in its genome afertility-restoring gene linked to a “small seed” phenotype marker, b)genotyping the seeds obtained by means of the cross according to stepa), c) self-fertilizing the maize plants derived from the seedsgenotyped according to step b), d) selecting the seeds homozygous forthe AMS transgene and heterozygous for the fertility-restoring genelinked to a “small seed” phenotype marker.
 3. A maize seed homozygousfor an AMS transgene and heterozygous for a fertility-restoring genelinked to a “small seed” phenotype marker, which can be obtained by themethod as claimed in claim
 1. 4. A method for the production of maizeseeds homozygous for a transgene conferring artificial nuclear malesterility (“AMS”), comprising the steps consisting in: a) crossing amale sterile maize plant heterozygous for the AMS transgene with afertility-restoring maize plant comprising in its genome afertility-restoring gene linked to a “small seed” phenotype marker, b)selecting, by means of the “small seed” phenotype, the maize seedscomprising in their genome a fertility-restoring gene linked to a “smallseed” phenotype marker, c) self-fertilizing the maize plants derivedfrom the seeds selected according to step b), d) selecting seedshomozygous for the AMS transgene and heterozygous for thefertility-restoring gene linked to a “small seed” phenotype marker, e)self-fertilizing maize plants derived from seeds according to step d),f) selecting seeds homozygous for the AMS transgene.
 5. A method for theproduction of maize seeds homozygous for a transgene conferringartificial nuclear male sterility (“AMS”), comprising the stepsconsisting in: a) crossing a male sterile maize plant heterozygous forthe AMS transgene with a fertility-restoring maize plant comprising inits genome a fertility-restoring gene linked to a “small seed” phenotypemarker, b) genotyping the seeds obtained by means of the cross accordingto step a), c) self-fertilizing the maize plants derived from the seedsgenotyped according to step b), d) selecting the seeds homozygous forthe AMS transgene and heterozygous for the fertility-restoring genelinked to a “small seed” phenotype marker, e) self-fertilizing maizeplants derived from seeds according to step d), f) selecting seedshomozygous for the AMS transgene.
 6. A method for the production ofmaize seeds homozygous for an AMS transgene, comprising the stepsconsisting in: a) self-fertilizing maize plants derived from seeds asclaimed in claim 3, b) selecting seeds homozygous for an AMS transgene.7. The method as claimed in claim 1, characterized in that at least oneselection step comprises densimetric separation.
 8. The method asclaimed in claim 7, characterized in that the densimetric separation iscarried out using a densimetric table.
 9. A method for the production ofa seed heterozygous for an AMS transgene, comprising the crossing of amaize plant derived from a seed homozygous for an AMS transgene, whichcan be obtained by the method as claimed in claim 4, with a maize planthaving a wild-type genotype.
 10. A method for the production of a seedheterozygous for an AMS transgene, characterized in that the method asclaimed in claim 4 also comprises the crossing of a maize plant derivedfrom said seed homozygous for an AMS transgene, with a maize planthaving a wild-type genotype.
 11. The method as claimed in claim 1, inwhich the AMS transgene conferring artificial nuclear male sterility isthe barnase gene, which is included in an expression cassette, under thecontrol of a promoter specific for pollen formation, in particular ananther-specific promoter such as pA3, pA6, pA9, pTA29, or of the Mac2promoter, and of the CaMV 3′ or Nos 3′ terminator, genetically linked toa gene encoding a selection agent under the control of the actinpromoter-actin intron and of the CaMV 3′ or Nos 3′ terminator.
 12. Themethod as claimed in claim 11, characterized in that the expressioncassette comprising the barnase gene also comprises a gene encoding aprotein of therapeutic and/or prophylactic interest genetically linkedto the barnase gene.
 13. The method as claimed in claim 11,characterized in that said promoter is the pA9 promoter specific forpollen formation.
 14. The method as claimed in claim 11, characterizedin that said gene encoding a selection agent is chosen from the bar genewhich confers resistance to the herbicide Basta® and the NptII genewhich confers resistance to kanamycin, said gene being included withinthe Ds transposable element.
 15. An expression cassette comprising afertility-restoring gene genetically linked to at least one geneencoding a “small seed” phenotype, combined with elements which allowtheir expression in plant cells, in particular a transcription promoterand terminator.
 16. The expression cassette as claimed in claim 15,characterized in that said fertility-restoring gene is the barstar geneplaced under the control of a promoter specific for pollen formation, inparticular an anther-specific promoter such as pA3, pA6, pA9, pTA29, orof the Mac2 promoter, and of the CaMV 3′ or Nos 3′ terminator,genetically linked to a gene encoding a selection agent under thecontrol of the actin promoter-actin intron and of the CaMV 3′ or Nos 3′terminator.
 17. The expression cassette as claimed in claim 15,characterized in that said gene encoding a “small seed” phenotype ischosen from the shrunken 2 and brittle 2 genes in antisense orientation.18. The expression cassette as claimed in claim 15, characterized inthat the promoter combined with the gene encoding a “small seed”phenotype is chosen from the HMWG and B32 promoters.
 19. The expressioncassette as claimed in claim 15, characterized in that said terminatoris chosen from the Nos 3′ terminator and the CaMV 3′ terminator.
 20. Avector, in particular a plasmid, characterized in that it contains atleast one expression cassette as described in claim
 11. 21. A cellularhost, in particular a bacterium such as Agrobacterium tumefacienstransformed with a vector as claimed in claim
 20. 22. A maize celltransformed with at least one vector as claimed in claim
 20. 23. Afertility-restoring maize plant, characterized in that it comprises inits genome a fertility-restoring gene linked to a “small seed” phenotypemarker.
 24. A maize plant homozygous for an AMS transgene andheterozygous for a fertility-restoring gene linked to a “small seed”phenotype marker, obtained from a seed as claimed in claim
 3. 25. Amethod for the multiplication of a maize plant homozygous for an AMStransgene and heterozygous for a fertility-restoring gene linked to a“small seed” phenotype marker, comprising the steps consisting in: a)self-fertilizing maize plants homozygous for an AMS transgene andheterozygous for a fertility-restoring gene linked to a “small seed”phenotype marker, which can be obtained by the method as claimed inclaim 1, b) selecting seeds homozygous for the AMS transgene and havinga “small seed” phenotype, c) selecting the seeds homozygous for the AMStransgene and heterozygous for a fertility-restoring gene linked to a“small seed” phenotype marker, obtained by self-fertilization of themaize plants obtained from the seeds obtained according to step b). 26.The method as claimed in claim 25, characterized in that step b)comprises densimetric separation.
 27. A kit for implementing the methodas claimed in claim 25, characterized in that it comprises maize seedshomozygous for an AMS transgene and heterozygous for afertility-restoring gene linked to a “small seed” phenotype marker, andoligonucleotides specific for the AMS transgene that are useful asprimers for detecting, by PCR, the seeds homozygous for an AMS transgeneand heterozygous for a fertility-restoring gene linked to a “small seed”phenotype marker.
 28. A maize seed homozygous for an AMS transgene andheterozygous for a fertility-restoring gene linked to a “small seed”phenotype marker, which can be obtained by the method as claimed inclaim
 2. 29. A method for the production of maize seeds homozygous foran AMS transgene, comprising the steps consisting in: a)self-fertilizing maize plants derived from seeds as claimed in claim 28,b) selecting seeds homozygous for an AMS transgene.
 30. A vector, inparticular a plasmid, characterized in that it contains at least oneexpression cassette as described in claim
 15. 31. A cellular host, inparticular a bacterium such as Agrobacterium tumefaciens transformedwith a vector as claimed in claim
 30. 32. A maize cell transformed withat least one vector as claimed in claim 30.